Targeting -dependent 9 and myeloid cell leukaemia 1 in MYC-driven B-cell lymphoma

Gareth Peter Gregory ORCID ID: 0000-0002-4170-0682

Thesis for Doctor of Philosophy

September 2016

Sir Peter MacCallum Department of Oncology The University of Melbourne

Doctor of Philosophy Submitted in total fulfilment of the degree of

Abstract

Aggressive B-cell lymphomas include diffuse large B-cell lymphoma, Burkitt lymphoma and intermediate forms. Despite high response rates to conventional immuno-chemotherapeutic approaches, an unmet need for novel therapeutic by resistance to chemotherapy and radiotherapy. The proto-oncogene MYC is strategies is required in the setting of relapsed and refractory disease, typified frequently dysregulated in the aggressive B-cell lymphomas, however, it has proven an elusive direct therapeutic target.

MYC-dysregulated disease maintains a ‘transcriptionally-addicted’ state, whereby perturbation of A significant body of evidence is accumulating to suggest that RNA polymerase II activity may indirectly antagonise MYC activity. Furthermore, very recent studies implicate anti-apoptotic myeloid cell leukaemia 1 (MCL-1) as a critical survival determinant of MYC-driven lymphoma.

This thesis utilises pharmacologic and genetic techniques in MYC-driven models of aggressive B-cell lymphoma to demonstrate that cyclin-dependent kinase 9 (CDK9) and MCL-1 are oncogenic dependencies of this subset of disease. The cyclin-dependent kinase inhibitor, dinaciclib, and more selective CDK9 inhibitors downregulation of MCL1 are used to demonstrate efficient induction conferred at least in part by other transcriptional cyclin-dependent that are required for viability of transcription. Furthermore, a genetic screen identifies MYC-driven lymphoid disease.

Finally, having established MCL-1 as a critical oncogenic dependency of MYC-driven direct pharmacologic antagonism of MCL-1 using a small molecule BH3-mimetic lymphoma, this thesis demonstrates the significant activity that is conferred by dependency involving CDK9 regulated RNA polymerase II-mediated transcription inhibitor of MCL-1. These findings confirm a druggable pathway of oncogenic cMYC of MCL-1, and proposes pharmacologic inhibition of CDK9 and MCL-1 as novel anti-lymphoma strategies.

i Declaration

This is to certify that:

1. This thesis comprises only my original work towards the degree of Doctor of Philosophy except where indicated in the preface. 2. Due acknowledgement has been made in the text to all other material used. 3. The thesis is fewer than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices.

Gareth Gregory 24 September 2016

ii Preface

The following generous contributions of experimental work depicted in this thesis are acknowledged:

Figure 3.4 Lymphoma transplantation performed by Ms Eva Vidacs

Figure 4.8 Real-time PCR assisted by Mr Joshua Hilton

Figure 4.11 Mouse retro-orbital bleeds performed by Ms Eva Vidacs

Figure 4.15 Experiment performed in conjunction with Mr Simon Hogg

Figures 4.17-4.19 Experiments performed in conjunction with Mr Zheng Fan

Figures 5.11-5.12 AZ-MCL1 administered by Ms Eva Vidacs

Gregory GP, Hogg SJ, Kats LM, Vidacs E, Baker AJ, Gilan O, Lefebure M, Martin BP, Dawson MA, Johnstone RW, Shortt J. 2015. CDK9 inhibition by dinaciclib potently suppresses Mcl-1 to induce durable apoptotic responses in aggressive MYC-driven B-cell lymphoma in vivo. Leukemia 29:1437-41.

Figure 1c Experiment performed in conjunction with Mr Simon Hogg

Figure 1d Experiment assisted by Dr Omer Gilan

Figures 2a-d Lymphoma transplantation performed by Ms Eva Vidacs

iii Acknowledgements

I would like to acknowledge the incomparable supervision and mentoring provided by my doctorate supervisors, Professor Ricky Johnstone and Associate Professor Jake Shortt. Beyond their supervision, they have fostered an environment in which I have been inspired and nurtured to thrive as a Clinician-Scientist and I value the ongoing roles that they will play in my path beyond the scope of these studies. Furthermore, I would like to thank my research mentor, Professor Joe Trapani, and the incredible training, assistance and support offered by my colleagues in the Johnstone laboratory. Particular mention must go to Ben Martin, Eva Vidacs, Marcus Lefebure, Simon Hogg, Lev Kats, Leonie Cluse, Joshua Hilton, Zheng Fan, core facilities. Research was funded by the Leukaemia Foundation of Australia, the Izabela Todorovski, Linda Stevens and members of the animal and flow cytometry Royal Australasian College of Physicians and Cancer Therapeutics CRC.

I would also like to acknowledge the extraordinary contributions from my family: the patience and support offered by my wife Amy and the unquestionable support and opportunities provided by my parents, Peter and Sylvia. The pathway toward these doctoral studies was inspired by a number of Haematologists and Clinician- Researchers who fostered a supportive environment for pursuing this career. I would like to acknowledge Drs Paul Cannell, Matthew Wright and Julian Cooney (Royal Perth Hospital); Professors Hatem Salem and Andrew Spencer (Alfred Health & Monash University); and Dr George Grigoriadis (Monash Health and Monash University) for their advice and support toward pursuing a research pathway in haematology.

Finally, I would like to acknowledge the incredible support, mentoring and opportunities offered by Associate Professor Stephen Opat (Monash Health & Monash University). Beyond all of these roles, Stephen, Jake and Ricky were instrumental in ensuring that the preclinical promise of dinaciclib described in studies herein was rapidly translated into an international phase I clinical trial and furthermore offered and encouraged me to take the role of lead investigator on this trial. I am indebted to them for such an opportunity.

iv Publications arising from this thesis

Gregory GP, Hogg SJ, Kats LM, Vidacs E, Baker AJ, Gilan O, Lefebure M, Martin BP, Dawson MA, Johnstone RW, Shortt J. 2015. CDK9 inhibition by dinaciclib potently suppresses Mcl-1 to induce durable apoptotic responses in aggressive MYC-driven B-cell lymphoma in vivo. Leukemia 29:1437-41.

Waibel W, Gregory G, Shortt J, Johnstone RW. Rational combination therapies targeting survival signaling in aggressive B cell leukemia / lymphoma. 2014. Curr Opin Hematol. 21(4):297-308.

Baker AJ, Gregory GP, Verbrugge I, Kats L, Hilton JJ, Vidacs E, Lee EM, Locke RB, Zuber J, Shortt J, Johnstone RJ. 2016. Apoptotic and therapeutic effects of dinaciclib in pre-clinical models of MLL-rearranged acute myeloid leukemia. Cancer Res 76(5):1158-69.

Hogg SJ, Newbold A, Martin BP, Gregory GP, Lefebure M, Vidacs E, Tothill R, Bradner JE, Shortt J, Johnstone RW. 2016. BET-inhibition induces apoptosis in aggressive B-cell lymphoma via epigenetic regulation of BCL-2 family members. Mol Cancer Ther. 15(9):2030-41.

Lefebure M, Tothill RW, Kruse E, Hawkins E, Shortt J, Mathews G, Gregory GP, Martin BP, Kelly MJ, Todorovski I, Doyle MA, Lupat R, Li J, Schroeder J, Wall M, Craig S, Poortinga G, Cameron D, Bywater M, Kats L, Gearhart MD, Bardwell V, Dickins RA, Hannan RD, Papenfuss T, Johnstone RW. Genomic characterisation of Eµ-Myc Bcor as a MYC co-operative tumor-suppressor [revision under review]. lymphomas by massively-parallel sequencing identifies

v Table of Contents

Abstract...... i Declaration...... ii Preface...... iii Acknowledgements...... iv Publications arising from this thesis...... v Table of Contents...... vi Table of Tables...... xiv Table of Figures...... xv Abbreviations...... xix

Chapter 1: The role of cyclin-dependent kinases and apoptosis family members in MYC-driven lymphoid malignancy...... 1

1.1 MYC-driven B-cell lymphoma...... 2 1.1.1 Conventional therapeutic approaches to aggressive B-cell lymphoma...... 2 1.1.2 Current novel therapeutic approaches to aggressive B-cell lymphoma...... 3 1.1.2.1 Chronic active BCR signalling...... 6 1.1.2.1.1 Targeting chronic active BCR signalling...... 6 1.1.2.2 Tonic BCR signalling...... 7 1.1.2.2.1 Targeting tonic BCR signalling...... 8 1.1.2.3 Non-BCR targeted approaches...... 8 1.1.2.3.1 EZH2 inhibition...... 8 1.1.2.3.2 BCL-6 inhibition...... 8 1.1.2.3.3 HDAC inhibition...... 9 1.1.2.3.4 BET-bromodomain inhibition...... 9 1.1.2.3.5 BCL-2 inhibition...... 10 1.1.2.3.6 RNA polymerase I inhibition...... 10 1.1.2.3.7 Immunological approaches...... 10 1.1.2.3.7.1 PD-1 / PD-L1 Checkpoint inhibition...... 10 1.1.2.3.7.2 Type II CD20 monoclonal antibodies...... 11 1.1.2.3.7.3 CD38 monoclonal antibodies...... 12 1.1.2.3.7.4 Radioconjugates...... 12

vi 1.1.2.3.7.5 13 1.1.2.3.7.6 CAR-T cells...... 13 Bi-specific T-cell engagers...... 1.1.2.4 Summary of current novel approaches...... 13 1.1.3 Murine modelling of MYC-driven B-cell lymphoma...... 14 1.1.3.1 The Eµ-Myc model...... 14 1.1.3.2 Other spontaneous murine models of MYC-driven B-cell lymphoma...... 15 1.1.3.3 Xenografting human MYC-driven B-cell lymphoma...... 15 1.2 MYC...... 15 1.2.1 MYC forms, structure and function...... 15 1.2.2 Regulation of MYC...... 16 1.2.3 Dysregulated MYC activity in cancer...... 18 1.2.4 MYC and positive transcription elongation factor B...... 18 1.2.5 RNA polymerase II activation and termination...... 19 1.3 Cyclin-dependent kinases...... 22 1.3.1 Transcriptional cyclin-dependent kinases...... 24 1.3.1.1 CDK7...... 24 1.3.1.2 CDK8...... 26 1.3.1.3 CDK9...... 26 1.3.1.4 CDK10...... 28 1.3.1.5 CDK11...... 28 1.3.1.6 CDK12...... 29 1.3.1.7 CDK13...... 30 1.3.1.8 CDK19...... 30 1.3.1.9 CDK20...... 30 1.3.2 Non-transcriptional cyclin-dependent kinases...... 31 1.3.2.1 CDK1...... 31 1.3.2.2 CDK2...... 32 1.3.2.3 CDK3...... 32 1.3.2.4 CDK4...... 33 1.3.2.5 CDK5...... 33 1.3.2.6 CDK6...... 34 1.3.2.7 CDK14...... 34 1.3.2.8 CDK15...... 34

vii 1.3.2.9 CDK16...... 35 1.3.2.10 CDK17...... 35 1.3.2.11 CDK18...... 36 1.3.3 ...... 36 1.3.4 Cyclin-dependent kinase-like family...... 36 1.3.5 Cyclin-dependent kinase inhibitors...... 37 1.3.5.1 Flavopiridol...... 37 1.3.5.2 Seliciclib...... 37 1.3.5.3 Dinaciclib...... 39 1.3.5.4 Palbociclib...... 40 1.3.5.5 Other cyclin-dependent kinase inhibitors...... 40 1.4 Apoptosis...... 41 1.4.1 Intrinsic apoptosis...... 41 1.4.1.1 Effector ...... 43 1.4.1.1.1 BAK...... 45 1.4.1.1.2 BAX...... 45 1.4.1.1.3 BOK...... 46 1.4.1.2 Anti-apoptotic proteins...... 46 1.4.1.2.1 BCL-2...... 47 1.4.1.2.2 MCL-1...... 48

1.4.1.2.3 BCL-XL...... 50 1.4.1.2.4 A1...... 51 1.4.1.2.5 BCL-W...... 51 1.4.1.2.6 BCL-B...... 51 1.4.1.3 BH3-only proteins...... 51 1.4.1.3.1 BIM...... 52 1.4.1.3.2 BID...... 52 1.4.1.3.3 PUMA...... 52 1.4.1.3.4 NOXA...... 53 1.4.1.3.5 BAD...... 53 1.4.1.3.6 BMF...... 53 1.4.1.3.7 BIK...... 54 1.4.1.3.8 HRK...... 54 1.4.1.4 Downstream effectors of apoptosis induction...... 54

viii 1.4.2 Extrinsic apoptosis...... 55 1.4.3 Small molecule inhibition of BCL-2 family members...... 55 1.5 Conclusions and hypothesis...... 56

Chapter 2: Materials & Methods...... 59

2.1 Tissue culture...... 60 2.1.1 Eµ-Myc lymphoma cell culture...... 60 2.1.1.1 60 2.1.1.2 Eµ- Media (EMM)...... 60 AnneMyc Kelso Modified DMEM...... 2.1.2 Human IG-MYC-translocated cell culture...... 60 2.1.3 Cell lines for retroviral and lentiviral use...... 62 2.1.4 Compounds for in vitro use...... 62 2.2 Flow cytometry apoptosis and assays...... 62 2.2.1 Cell Death assays...... 62 2.2.1.1 Annexin-V and propidium iodide apoptosis assays...... 64 2.2.1.2 Mitochondrial outer membrane potential assessment (TMRE)...... 64 2.2.1.3 Cell cycle analysis according to nuclear DNA content..... 64 2.3 techniques...... 64 2.3.1 Preparation of protein...... 64 2.3.2 Protein separation, transfer and immunoblotting...... 65 2.4 Ribonucleic acid techniques...... 67 2.4.1 Preparation of RNA...... 67 2.4.2 Generation of complementary DNA...... 67 2.4.3 Quantitative reverse-transcription PCR...... 67 2.4.3.1 qRT-PCR statistical analysis...... 68 2.5 Chromatin immunoprecipitation...... 68 2.5.1 Preparation of immunoprecipitated samples...... 68 2.5.2 Separation, extraction and quantitation of DNA...... 70 2.6 Retroviral transduction...... 70 2.6.1 Retroviral supernatant production...... 70 2.6.1.1 Retroviral transduction of Eµ-Myc lymphoma cells...... 70 2.6.2 Lentiviral transduction...... 71 2.6.2.1 Lentiviral supernatant production...... 71

ix 2.6.2.2 Lentiviral transduction of OPM2 cells...... 71 2.6.3 Cloning shRNA into inducible systems...... 74 2.6.4 Competitive cell growth/proliferation assays...... 74 2.6.4.1 Competitive assays of retrovirus-transduced cells...... 74 2.6.4.2 Competitive assays of lentivirus-transduced cells...... 75 2.7 In vivo experimentation...... 75 2.7.1 Experimental animals and housing...... 75 2.7.1.1 Preparation of therapeutics for in vivo administration.. 75 2.7.1.1.1 A1592668.1...... 77 2.7.1.1.2 ABT-199...... 77 2.7.1.1.3 AZ-MCL1...... 77 2.7.1.1.4 AZD4320...... 77 2.7.1.1.5 Dinaciclib...... 78 2.7.1.1.6 Fedratinib...... 78 2.7.1.1.7 Ibrutinib...... 78 2.7.1.2 Administration of therapeutics...... 78 2.7.1.3 Assessment of experimental animals...... 78 2.7.2 In vivo experimentation...... 79 2.7.2.1 Transplantation of Eµ-Myc lymphoma...... 79 2.7.2.1.1 Assessment of Full Blood Examination (FBE) and lymphoma burden in blood according to number and GFP representation...... 79 2.7.2.1.2 In vivo apoptosis assays using Eµ-Myc lymphoma...... 79 2.7.2.2 Myc myeloma...... 80 2.7.2.3 Transplantation of Burkitt lymphoma cell line...... 80 Transplantation of Vκ* 2.7.3 Histology of in vivo-derived specimens...... 80 2.7.3.1 Hematoxylin and eosin stain...... 80 2.7.3.2 Terminal deoxynucleotidyl dUTP nick-end labelling (TUNEL)...... 80 2.7.4 Biochemistry assessment...... 81 2.7.4.1 Serum protein electrophoresis (SPEP)...... 81 2.7.4.2 Serum liver, pancreatic and muscle assessment...... 81 2.7.5 In vivo imaging...... 81 2.8 Statistical analyses...... 82

x Chapter 3: In vitro and in vivo characterisation of CDK9 inhibition with dinaciclib as an effective therapeutic strategy for MYC-driven B-cell lymphoma...... 83

3.1 Introduction...... 84 3.2 Results...... 87 3.2.1 Dinaciclib treatment potently induces apoptosis of Eµ-Myc lymphoma and human IG-cMYC-translocated lymphoma in vitro...... 87 3.2.2 Dinaciclib treatment of Eµ-Myc lymphoma leads to CDK9 inhibition of RNA polymerase II activation and reduced Mcl-1 protein expression...... 91 3.2.3 Dinaciclib therapy is associated with CDK9 inhibitory activity and prolonged survival of tumour-bearing mice in vivo...... 91 3.2.4 CDK9 inhibition by dinaciclib potently suppresses Mcl- 1 to induce durable apoptotic responses in aggressive MYC-driven B-cell lymphoma in vivo...... 102 3.3 Discussion...... 114 3.3.1 Dinaciclib as a potent inhibitor of Pol II activation...... 114 3.3.2 Dinaciclib potently induces apoptosis of MYC-driven B-cell lymphoma...... 114 3.3.3 Dinaciclib is a tolerable and effective therapy against MYC-driven B-cell lymphoma in vivo...... 117 3.3.4 Dinaciclib effects on MYC expression...... 117 3.3.5 Conclusion...... 118

Chapter 4: Genetic and pharmacologic targeting of CDK9 in MYC-driven models of B-cell lymphoma...... 119

4.1 Introduction...... 120 4.2 Results...... 122 4.2.1 CDK9 inhibitors potently induce apoptosis of Eµ- Myc lymphoma and human IG-cMYC-translocated lymphoma in vitro...... 122 4.2.2 CDK9-inhibitor treatment of Eµ-Myc lymphoma leads to inhibition of RNA polymerase II activation and reduced Mcl-1 expression...... 134 4.2.3 CDK9-inhibitor therapy is associated with prolonged survival of tumour-bearing mice in vivo...... 135

xi 4.2.4 CDK9-inhibitor-mediated repression of Mcl-1 and inhibition of Bcl-2 display additivity in vitro and synergise in vivo in MYC-translocated lymphoid malignancy...... 146 4.2.5 Genetic depletion of Cdk9 is detrimental to Eµ-Myc lymphoma...... 156 4.2.6 novel CDKs which are critical to MYC-driven lymphoid malignancy...... A boutique CDK RNA-interference screen identifies 157 4.3 Discussion...... 170 4.3.1 CDK9 inhibitors as potent inhibitors of Pol II activation...... 170 4.3.2 CDK9 inhibitors potently induce apoptosis of MYC- driven B-cell lymphoma...... 170 4.3.3 CDK9 inhibitors confer differing effects upon cell cycle progression...... 171 4.3.4 CDK9 inhibitors confer differing effects upon Myc transcription...... 171 4.3.5 CDK9 inhibition represents a tolerable and effective therapy against MYC-driven B-cell lymphoma in vivo..... 173 4.3.6 therapeutic targets in MYC-driven lymphoid malignancies...... Transcriptional kinases represent bona fide 174 4.3.7 Bcl-2 antagonism accelerates MYC-driven lymphoma progression...... 174 4.3.8 Conclusion...... 176

Chapter 5: Genetic and pharmacologic targeting of MCL-1 in MYC-driven models of B-cell lymphoma...... 177

5.1 Introduction...... 178 5.2 Results...... 180 5.2.1 MCL-1 inhibition potently induces apoptosis of Eµ- Myc lymphoma and human IG-cMYC-translocated lymphoma in vitro...... 180 5.2.2 MCL-1-inhibitor treatment of Eµ-Myc lymphoma is associated with biomarkers of rapid intrinsic apoptosis induction...... 186 5.2.3 MCL-1-inhibitor therapy is tolerated and induces apoptosis of Eµ-Myc lymphoma in vivo...... 187 5.2.4 MCL-1 inhibitor therapy is associated with prolonged survival of tumour-bearing mice in vivo...... 187 5.2.5 Genetic depletion of Mcl-1 is detrimental to MYC- driven lymphoma...... 197 xii 5.3 Discussion...... 205 5.3.1 MCL-1 inhibition potently induces apoptosis of MYC- driven B-cell lymphoma...... 205 5.3.2 MCL-1 inhibition represents a tolerable and effective therapy against MYC-driven B-cell lymphoma in vivo..... 206 5.3.3 Conclusions...... 207

Chapter 6: Summation and Conclusions...... 209

6.1 The MYC / CDK9 / Pol II / MCL-1 axis as an oncogenic pathway...... 210 6.2 Dinaciclib demonstrates marked anti-lymphoma activity...... 210 6.2.1 CDK9 inhibitors demonstrate marked anti-lymphoma activity...... 211 6.2.2 Targeting transcriptional addiction in MYC-driven lymphoma...... 211 6.3 MCL-1 inhibition demonstrates marked anti- lymphoma activity...... 212 6.4 Translation of CDK9 inhibitors to the clinic...... 212 6.5 Conclusions...... 213

Chapter 7: Bibliography...... 215

xiii Table of Tables

Chapter 1: The role of cyclin-dependent kinases and apoptosis family members in MYC-driven lymphoid malignancy...... 1

Table 1.1: 38

Chapter 2: MaterialsInhibitory profilesand methods...... of CDK inhibitors...... 59

Table 2.1: Characteristics of Eµ-Myc Myc multiple myeloma used in experimental work...... 61 Table 2.2: Chemical compounds described lymphomas in in vitro and Vκ*studies...... 63 Table 2.3: Primary and secondary antibodies...... 66 Table 2.4: Primer sequences used for qRT-PCR and ChIP experimentation...... 69 Table 2.5: Lentiviral construct sequences...... 72 Table 2.6: Short-hairpin RNA sequences targeting Cdk9 and Mcl1...... 73 Table 2.7: Therapeutics used for in vivo studies...... 76

Chapter 5: Genetic and pharmacologic targeting of MCL-1 in MYC-driven models of B-cell lymphoma...... 177

Table 5.1: 181

Inhibitory profile of the MCL-1 inhibitor AZ-MCL1......

xiv Table of Figures

Chapter 1: The role of cyclin-dependent kinases and apoptosis family members in MYC-driven lymphoid malignancy...... 1

Figure 1.1: Chronic active and tonic B-cell signalling pathways...... 4 Figure 1.2: MYC interactions with apoptosis pathways...... 17 Figure 1.3: CDK7 and CDK9 regulation of Pol II pause-release and productive elongation...... 20 Figure 1.4: Interaction of CDKs with the cell cycle...... 23 Figure 1.5: Subgrouping of CDKs and their cognate cyclin binding 25 partners...... Figure 1.6: Intrinsic apoptosis pathway...... 42 Figure 1.7: Protein structure of BCL-2 family members...... 44 Figure 1.8: Proposed ‘linear pathway’ involving CDK9 regulation of Pol II-mediated transcription of critical MYC targets including MCL-1...... 57

Chapter 3: In vitro and in vivo characterisation of CDK9 inhibition with dinaciclib as an effective therapeutic strategy for MYC-driven B-cell lymphoma...... 83

Figure 3.1: CDK9 regulates full activation of Pol II-mediated transcription of critical MYC targets including MCL-1.... 85 Figure 3.2: Dinaciclib induces apoptosis of Eµ-Myc and human IG- cMYC-translocated lymphoma in vitro...... 88 Figure 3.3: Dinaciclib treatment is associated with cytostasis and apoptosis of Eµ-Myc lymphoma in vitro...... 90 Figure 3.4: Dinaciclib treatment is associated with reduced Pol II activation and Mcl-1 protein expression in apoptosis- protected lymphoma cells...... 92 Figure 3.5: Dinaciclib treatment is associated with reduced Pol II activation and Mcl-1 protein expression in wild-type lymphoma cells...... 94 Figure 3.6: Dinaciclib therapy is associated with reduced Pol II activation and Mcl-1 protein expression in vivo...... 97 Figure 3.7: Dinaciclib therapy is associated with biomarkers of disease response and prolongation of overall survival in vivo...... 98 Figure 3.8: Comparative overall survival conferred by other targeted therapies against Eµ-Myc lymphoma in vivo..... 100

xv Figure 3.9: CDK9-inhibition with dinaciclib reduces Pol II- mediated transcription of critical MYC targets including MCL-1...... 115

Chapter 4: Genetic and pharmacologic targeting of CDK9 in MYC-driven models of B-cell lymphoma...... 119

Figure 4.1: CDK9 regulates full activation of Pol II-mediated transcription of critical MYC targets including MCL-1.... 121 Figure 4.2: Flavopiridol induces apoptosis of Eµ-Myc lymphoma in vitro...... 124 Figure 4.3: A selective CDK4/9 inhibitor induces apoptosis of Eµ- Myc lymphoma in vitro...... 126 Figure 4.4: The selective CDK4/6 inhibitor palbociclib does not induce apoptosis of Eµ-Myc lymphoma in vitro...... 128 Figure 4.5: The selective CDK9 inhibitor AZ-CDK9 induces apoptosis of Eµ-Myc lymphoma in vitro...... 130 Figure 4.6: Pharmacologic inhibition of CDK9 is associated with cytostasis of apoptosis-protected Eµ-Myc lymphoma in vitro...... 132 Figure 4.7: Pharmacologic inhibition of CDK9 is associated with apoptosis of human IG-MYC-translocated lymphoma and myeloma cell lines in vitro...... 136 Figure 4.8: Pharmacologic inhibition of CDK9 is associated with repression of MCL-1 transcription in vitro...... 138 Figure 4.9: CDK9-inhibitor treatment is associated with reduced Pol II activation and Mcl-1 protein expression in MYC- driven lymphoma cells...... 140 Figure 4.10: CDK9 inhibitor therapy is associated with reduced Pol II activation and Mcl-1 protein expression in vivo...... 142 Figure 4.11: CDK9 inhibitor therapy is associated delayed disease progression and prolongation of overall survival in vivo...... 144 Figure 4.12: Pharmacologic inhibition of Bcl-2 does not induce Myc lymphoma at on-target concentrations in vitro...... 148 Figure 4.13: Pharmacologicapoptosis of Eμ- inhibition of Bcl-2 does not synergise Myc lymphoma in vitro...... 150 Figure 4.14: Pharmacologicwith CDK9 inhibition inhibition to induce of Bcl-2 apoptosis in combination of Eμ- with CDK9 inhibition is associated with prolonged survival of Eµ-Myc tumour-bearing mice in vivo...... 152 Figure 4.15: Combination pharmacologic inhibition of Bcl-2 and Myc tumour-bearing mice in vivo...... 154 CDK9 is associated with prolonged survival of Vκ* xvi Figure 4.16: Genetic depletion of Cdk9 with shRNA phenocopies CDK9 inhibition of Eµ-Myc lymphoma...... 158 Figure 4.17: Preliminary boutique RNAi lentiviral CDK screen using OPM2 cells...... 162 Figure 4.18: Boutique RNAi lentiviral CDK screen using OPM2 cells

driven lymphoid neoplasia...... 164 Figure 4.19: Validationidentifies novel of positive oncogenic and negative dependencies results of from MYC- boutique RNAi lentiviral CDK screen...... 168 Figure 4.20: Model of proposed non-CDK9-mediated cytostatic activity of pharmacologic inhibitors of CDK9...... 172

Chapter 5: Genetic and pharmacologic targeting of MCL-1 in MYC-driven models of B-cell lymphoma...... 177

Figure 5.1: Targeting MCL-1 in a transcriptionally-addicted model of MYC-driven lymphoma...... 179

Figure 5.2: In vitro selectivity of MCL-1 and BCL-2 / BCL-XL inhibitors...... 182 Figure 5.3: In vitro selectivity of select MCL-1 inhibitor against a panel of derived Eµ-Myc cell lines...... 184 Figure 5.4: In vitro readouts of apoptosis induction for Eµ-Myc lymphoma upon pharmacologic MCL-1 inhibition...... 188

Figure 5.5: MCL-1 inhibition, but not BCL-2 / BCL-XL inhibition, is associated with apoptosis induction of human IG- cMYC-translocated lymphoid malignancy in vitro...... 190 Figure 5.6: AZ-MCL1 treatment is associated with biomarkers of rapid apoptosis induction...... 191 Figure 5.7: Selective MCL-1 inhibition is not associated with transcriptional changes to anti-apoptotic mRNA expression...... 192 Figure 5.8: AZ-MCL1 therapy in vivo is well tolerated systemically 193 Figure 5.9: AZ-MCL1 therapy in vivo is associated with selective apoptosisand associated of lymphoma with specific cells toxicity...... and biochemical evidence of organ toxicities...... 194 Figure 5.10: MCL-1 inhibitor therapy is associated with reduced disease progression and prolongation of survival for mice transplanted with Eµ-Myc lymphoma...... 198 Figure 5.11: MCL-1 inhibitor therapy is effective against null lymphoma in vivo...... 200 Figure 5.12: MCL-1 inhibitor therapy is associated with reduced disease progression and prolongation of survival for mice transplanted with human IG-cMYC-translocated Burkitt lymphoma...... 202

xvii Figure 5.13: Genetic depletion of Mcl-1 with shRNA leads to loss of representation of transduced lymphoma cells...... 204

xviii Abbreviations

-/- Null / knockout 2-ME 2-mercaptoethanol

A1 BCL-2-related protein A1 Ab Antibody ABC Activated B-cell ALT Alanine transaminase An Annexin-V AKT B ARF Alternate reading frame AST Aspartate aminotransferase

BAD BCL-2-associated death promoter BAK BCL-2 homologous antagonist / killer BAX BCL-2-associated X protein BCL-2 B-cell lymphoma 2 BCL-B BCL-2-like protein 10 BCL-W BCL-2-like protein 2 BCL-X BCL-2-like protein 1

BCL-XL B-cell lymphoma-extra large BCR B-cell receptor BET Bromodomain and extra-terminal domain

BH BCL-2 homology domain bHLHBFP BasicBlue fluorescent helix-loop-helix protein BAD BCL-2-associated death promoter BID BH3 interacting-domain death agonist BIK BCL-2-interacting killer BIM BCL-2-like protein 11 BMF BCL-2-modifying factor BOK BCL-2 related ovarian killer BSA Bovine serum albumin BTK Bruton tyrosine kinase

CAK CDK-activating kinase CDK Cyclin-dependent kinase ChIP Chromatin immunoprecipitation CK Creatine kinase CLL Chronic lymphocytic leukaemia CTD Carboxy-terminal domain

xix DLBCL Diffuse large B-cell lymphoma

DMSO Dimethylsulfoxide DNADMEM DeoxyribonucleicDulbecco’s modified acid Eagle medium DRB 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole DSIF DRB sensitivity-inducing factor

EDTA Ethylenediaminetetraacetic acid EMM Eµ-Myc media

FACS Fluorescence-activated cell sorter FBE Full blood examination FBS Fetal bovine serum FSC Forward scatter

G1 Growth phase 1 G2 Growth phase 2 GCB Germinal centre B-cell

GSK Glycogen synthase kinase GFP Green fluorescent protein HPBCD Hydroxypropyl-beta-cyclodextrin HPMC Hydroxypropylmethylcellulose HRK Activator of apoptosis harakiri HRP Horseradish peroxidase

IAP Inhibitor of apoptosis protein

IC50 Inhibitory concentration 50% IG Immunoglobulin IRES Internal ribosomal entry site

JAK Janus associated kinase kDa Kilodalton

LD50 Lethal dose 50% LZ Leucine zipper

M phase MAPK Mitogen activated protein kinase MCL-1 Myeloid cell leukaemia 1 MOM Mitochondrial outer membrane MOMP Mitochondrial outer membrane permeabilisation MSCV Murine virus MTD Maximum tolerated dose mTOR Mammalian target of rapamycin MYC Myelocytomatosis viral oncogene

xx NELF Negative elongation factor NFκB Nuclear factor kappa B NHL Non-Hodgkin lymphoma NOD Non-obese diabetic NOXA Phorbol-12-myristate-13-acetate-induced protein 1

P- Phospho- PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PCR Polymerase chain reaction PD-1 Programmed death 1 PEI Polyethylenimine PI Propidium iodide PI3K Phosphoinositide 3-kinase Pol II RNA polymerase II P-TEFb Positive transcription elongation factor B PUMA p53 upregulated modulator of apoptosis

Rb RNA Ribonucleic acid RNAi RNA interference Rpb1 RNA polymerase II subunit B1 RPM Revolutions per minute RT-PCR Reverse transcription polymerase chain reaction

S Synthesis phase

SDS Sodium dodecyl sulfate SECSCID SuperSevere elongation combined compleximmunodeficient SEM Standard error of the mean Ser Serine shRNA Short hairpin RNA / small hairpin RNA SPEP Serum protein electrophoresis SSC Side scatter

TGF-β Thr Threonine TMRE Tetramethylrhodamine,Transforming growth factor ethyl β ester, perchlorate TUNEL Terminal deoxynucleotidyltransferase-mediated nick-end labelling

UTR Untranslated region UV Ultraviolet

WBC White blood cell

XIAP X-linked IAP

µM Micromolar

xxi xxii Chapter 1: The role of cyclin-dependent kinases and apoptosis family members in MYC-driven lymphoid malignancy

1 Chapter 1

1.1 MYC-driven B-cell lymphoma

It is almost 60 years since Denis Burkitt first described the case series of young mandible (Burkitt 1958). We have since come to appreciate Burkitt lymphoma as African patients afflicted by an aggressive ‘sarcoma’ primarily affecting the perhaps the most aggressive of all cancers, characterised by a high proliferative rate as seen histologically with frequent mitotic and apoptotic cells and clinically by rapid symptomatic onset and disease progression (Burkitt 1958; Swerdlow

Burkitt lymphoma, mapping of the cMYC (MYC) et al. 2008). Following identification of recurrent cytogenetic translocations in its implication as the driving oncogene for initiation of Burkitt lymphomagenesis gene to 8 first led to (Dalla-Favera et al. 1982).

MYC-dysregulation is not, however, restricted to the prototypic disease Burkitt lymphoma. Indeed, MYC haematological and non-haematological cancers (Beroukhim et al. 2010). More has been shown to be frequently amplified across many recently, dysregulated MYC expression was reported in up to one-third of diffuse cytogenetic translocation of the MYC (Johnson et al. 2012b; Swerdlow et large B-cell lymphomas (DLBCL), despite a significantly lower proportion showing al. 2008). Together, Burkitt lymphoma, DLBCL and intermediate forms make up the spectrum of diseases informally referred to as aggressive B-cell lymphomas. Therefore, MYC-dysregulation is common to this spectrum of diseases, and may either directly or indirectly present alternative therapeutic targets in the treatment of these lymphomas.

1.1.1 Conventional therapeutic approaches to aggressive B-cell lymphoma

Despite a comprehensive understanding of the pathogenesis of Burkitt lymphoma including additional recurrent somatic mutational events, chemotherapy remains the mainstay of treatment. For younger, healthier patients in the developed world, intensive cytotoxic regimens involving combinations of cyclophosphamide, doxorubicin, vincristine, methotrexate, ifosfamide, etoposide and cytarabine (CODOX-M / IVAC, EPOCH) in combination with immunotherapy in the form of the CD20 monoclonal antibody, rituximab, have led to durable remissions and even cures for the majority of patients (Mead et al. 2002; Dunleavy et al. 2013). However, alternative treatment approaches are required for relapsed and refractory disease, supportive care infrastructure precludes their safe and effective use. as well as for patients unfit to tolerate these intensive regimens, or where a lack of

2 Chapter 1

The prognostic implication of dysregulated MYC expression or presence of MYC translocation to DLBCL remains unresolved. Elevated MYC expression or isolated MYC translocation has been shown to be associated with improved overall survival (Johnson et al. 2012b), though the authors report this may be an artefact of small numbers within that observational arm. In contrast, other groups have shown isolated MYC translocation (Savage et al. 2009; Copie-Bergman et al. 2015) or isolated high MYC protein expression (Perry et al. 2014) to associate with poorer prognosis. Poorest outcome is associated by presence of both MYC and BCL2 (B-cell lymphoma 2) translocation (double-hit lymphoma) or instances of concomitant high MYC and BCL-2 protein expression (Savage et al. 2009; Johnson et al. 2012b; Perry et al. 2014). The majority of published studies suggest MYC translocation or high protein expression to confer poor prognosis when compared to absence of translocation or low protein expression.

While standard immunochemotherapy with rituximab, cyclophosphamide,

(R-CHOP) remains the mainstay of therapy for DLBCL, recent evidence suggests doxorubicin, vincristine and prednisolone (Fisher et al. 1993; Coiffier et al. 2002) that infusional immunochemotherapy regimens such as rituximab, etoposide, prednisolone, vincristine, cyclophosphamide and doxorubicin (R-EPOCH) may associate with superior overall survival and progression free survival for poor risk disease including cytogenetic MYC / BCL2 translocated lymphoma (Oki et al. 2014). Again, these regimens are often not suitable due to unacceptable toxicity within the older population in which this disease is enriched. Therefore, there remains an unmet clinical need for novel therapeutic approaches within this disease group also.

1.1.2 Current novel therapeutic approaches to aggressive B-cell lymphoma

In order to describe the current novel therapeutic approaches for treatment of aberrations that confer oncogenic dependency upon different signalling proteins aggressive B-cell lymphoma in the clinic, one must first consider the signalling and Burkitt lymphoma cell lines and ex vivo patient tumour specimens has led and kinases. Pharmacologic and molecular genomic profiling of human DLBCL to the accepted paradigm of two discrete molecular subtypes of B-cell receptor (BCR)-driven oncogenic signalling; chronic active B-cell receptor signalling as is commonly associated with activated B-cell (ABC)-like DLBCL, and tonic B-cell receptor signalling, which is characteristic of Burkitt lymphoma and germinal centre B-cell-like (GCB) DLBCL (Figure 1.1) (Lenz et al. 2008; Davis et al. 2010; Sander et al. 2012; Schmitz et al. 2012; Shortt et al. 2013; Young and Staudt 2013).

3 Chapter 1

Figure 1.1: Chronic active and tonic B-cell receptor signalling pathways

(a) The chronic active B-cell receptor (BCR) signalling pathway. Antigenic stimulus of the BCR or activating mutation of nodes denoted in yellow are associated with activation of this pathway, leading to increased signalling through PI3K/AKT and NFκB pathways. Black arrows denote activation. Small molecule inhibitors (red text) are shown with black bars denoting direct inhibition; dashed bar denotes indirect inhibition. TLR, toll-like receptor. (b) The tonic BCR signalling pathway. Antigen-independent activation of this pathway leads to hyperactivation of the PI3K/AKT pathway. Gain of function mutation in TCF3 (yellow node) and loss of function mutation of ID3 (orange node) have been associated with activation of this pathway. Black arrows denote activation; black bars denote direct inhibition. Representative small molecule inhibitors are shown.

4 Chapter 1 a BCR TLR

Ibrutinib

A SYK BTK MYD88 CD79B CD79 Enzastaurin IRAK1 IRAK4

Fostamatinib PLC PKC

Idelalisib PI3K CARD11 BCL-10 MALT1

MK2206 AKT

NF B Lenalidomide Bortezomib

Everolimus mTOR BCL-2 Venetoclax b BCR

IgH

A

SHP-1 TCF3 CD79B

CD79 SYK

PI3K Idelalisib ID3

AKT MK2206

mTOR Everolimus

5 Chapter 1

Each of these distinct subtypes of oncogenic BCR signalling activation confers upregulation of differing downstream signalling proteins and kinases that present novel therapeutic targets for the respective disease subsets.

1.1.2.1 Chronic active BCR signalling

Normal B-cell proliferation and survival involves processes driven by BCR signalling (Kraus et al. 2004). Therefore, it is not surprising that malignant counterparts would exploit associated processes for oncogenesis. Chronic active BCR signalling is the process through which antigenic stimulus of the extracellular BCR components (immunoglobulin heavy, IgH, and light, IgV chains, respectively) and plasma membrane components CD79A and CD79B lead to activation of a linear intracellular signalling pathway involving internal tyrosine-based activation motifs (ITAMs), spleen tyrosine kinase (SYK), Bruton tyrosine kinase (BTK), domain-containing protein 11 (CARD11), B-cell CLL / lymphoma 10 (BCL-10) and gamma (PLCγ), beta (PKCβ), caspase recruitment mucosa-associated lymphoma translocation protein 1 (MALT1), with resultant activation of mitogen-activated protein kinase (MAPK), nuclear factor of activated T-cells (NFAT), / mammalian target of rapamycin (AKT/MTOR) Figure 1.1a) (Davis et al. 2010; Young and Staudt 2013). Hence, numerous potential molecular therapeutic targets are and nuclear factor κB (NFκB) pathways ( implicated in the chronic active BCR signalling pathway.

1.1.2.1.1 Targeting chronic active BCR signalling

clinical responses in the treatment of indolent B-cell lymphomas such as chronic Ibrutinib is the first-in-class inhibitor of BTK and has been associated with marked lymphocytic leukaemia (CLL) and mantle cell lymphoma (Byrd et al. 2013; Wang et al. 2013a). Ibrutinib monotherapy was associated with a number of clinical responses in a phase I/II study in relapsed and refractory ABC-type DLBCL (Wilson et al. 2015). Notably, this included responses in cases where the activating mutation was in myeloid differentiation primary response gene 88 (MYD88), downstream of

BTK, indicating that significant interplay between MYD88 and BCR pathways exists. CARD11 mutation, suggesting that activation of this downstream convergent point However, no responses were identified of the three lymphomas demonstrating likely renders insensitivity to BCR pathway antagonism. Collectively, these studies set the scene for current clinical trials assessing the combination of ibrutinib in combination with standard of care R-CHOP immunochemotherapy for treatment of ABC DLBCL (NCT01855750).

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immunomodulatory therapy with lenalidomide has been assessed preclinically As BCR signalling has been associated with NFκB pathway activation, production (Yang et al. 2012). Empiric addition of lenalidomide to R-CHOP has been and shown to augment lymphoma cell death through induction of interferon β shown to be well tolerated in a phase II study and overcome the typical negative prognostic impact of ABC DLBCL (Nowakowski et al. 2015). Similarly, chemotherapy has been shown to associate with an improvement in response inhibition with bortezomib counteracts NFκB activity and in combination with rate and overall survival for treatment of relapsed ABC, but not GCB, DLBCL (Dunleavy et al. 2009). Despite the requirement for SYK in linking BCR signalling to intracellular components of the pathway, a phase II clinical trial of the SYK inhibitor, fostamatinib, in relapsed and refractory DLBCL yielded a disappointing with enzastaurin in a similar patient population and disease group demonstrated overall response rate of only 3% (Flinn et al. 2016). However, direct PKCβ inhibition a number of durable complete and partial responses (Robertson et al. 2007). While many other BCR signalling molecules are currently subject to preclinical considered therapeutic options for ABC DLBCL. Contemporary updates on the therapeutic investigation, they are not yet sufficiently clinically advanced to be progress of these preclinical studies has been demonstrated in recent review articles by our group and others (Waibel et al. 2014; Young and Staudt 2013).

1.1.2.2 Tonic BCR signalling

Burkitt lymphoma and GCB DLBCL are not dependent upon chronic active BCR signalling. However, elegant studies have shown normal mature B cells to be which was only rescued by constitutive phosphoinositide 3-kinase (PI3K) significantly depleted following genetic deletion of CD79A or the ITAM, a process al. 1997; Srinivasan et al. 2009; Kraus et al. 2004). Furthermore, RNA sequencing expression, but not constitutive expression of NFκB pathway components (Lam et pathogenesis of Burkitt lymphoma, including 70% of analysed cases harbouring has identified recurrent somatic mutational events that are critical to the loss of function mutation of inhibitor of DNA binding 3 (ID3) or gain of function mutation of transcription factor 3 (TCF3) (Figure 1.1b) (Schmitz et al. 2012). ID3 is a binding repressor of TCF3, and TCF3 is involved in several cellular processes including repression of the BCR signalling inhibitor, Src homology region 2 domain- containing phosphatase-1 (SHP-1), and increasing transcription of the IGH and IGκ loci, leading to increased BCR expression (Schmitz et al. 2012). Hence, tonic BCR-dependent PI3K signalling involving PI3K, AKT and mTOR, together with profiling and sequencing of primary human lymphoma specimens 7 Chapter 1 provides the basis for molecular targets in mature B-cell lymphomas such as Burkitt lymphoma and GCB DLBCL (Figure 1.1b) (Sander et al. 2012; Schmitz et al. 2012; Love et al. 2012).

1.1.2.2.1 Targeting tonic BCR signalling

PI3K-related DNA damage response kinases in the management of MYC-driven Preclinical studies have shown efficacy of combined targeting of mTORC1 and B-cell lymphomas (Shortt et al. 2013). The mTOR inhibitor, everolimus, has

PI3K-inhibitor, copanlisib, is currently in clinical trial for the same disease group shown efficacy in a phase II study of relapsed aggressive lymphoma, and a novel

SYK inhibitor, R406, in potently killing multiple GCB DLBCL cell lines, although (Witzig et al. 2010). Furthermore, preclinical studies demonstrated efficacy of the the clinical SYK inhibitor, fostamatinib, showed disappointing results in the phase II clinical trial described in section 1.1.2.1.1 above (Chen et al. 2013; Flinn et al. 2016). A phase II study of the AKT inhibitor, MK2206, in relapsed DLBCL has been performed though no results have yet been presented (NCT01466868). Overall, in dependent lymphomas. these findings support the development of therapies targeting tonic BCR signalling

1.1.2.3 Non-BCR targeted approaches

1.1.2.3.1 EZH2 inhibition

Germinal centre lymphocytes are dependent upon activity of the histone methyltransferase, Enhancer of zeste homolog 2 (EZH2), and mouse modeling of EZH2 mutation has led to rapid germinal centre lympho-proliferation analogous to GCB DLBCL (Béguelin et al. 2013). Furthermore, gain of function mutations in EZH2 histone H3 lysine 27 (Morin et al. 2010; 2013). Hence, rational targeting of EZH2 are frequently identified in GCB DLBCL with resultant increased trimethylation of relapsed and refractory DLBCL (NCT02082977). with the specific inhibitor, tazemetostat, is the focus of a current clinical trial in

1.1.2.3.2 BCL-6 inhibition

repressor involved in the germinal centre reaction and its expression is restricted The zinc finger transcription factor B-cell lymphoma 6 (BCL-6) is a transcriptional to germinal centre B-lymphocytes (Swerdlow et al. 2008; Choi et al. 2009). Translocation or mutation of BCL6 occurs in up to 70% of historical DLBCL cohorts

8 Chapter 1 and associates with dysregulation of its expression, indicating that its targeting may represent a novel therapeutic approach (Ye et al. 1993; Migliazza et al. 1995). Topoisomerase II inhibition with etoposide chemotherapy has been shown to reduce BCL-6 expression and induce apoptosis of germinal centre-derived lymphomas in vitro (Kurosu et al. 2003). These studies provided rationale for use of etoposide-containing regimens such as dose-adjusted EPOCH for treatment of DLBCL, with younger patient groups with GCB-DLBCL showing favorable survival

(Wilson et al. 2012). Furthermore, preclinical studies of a direct BCL-6 inhibitor profiles and superior outcomes compared to comparators with ABC-DLBCL in vitro and in vivo, providing rationale for clinical development of BCL-6 inhibitors for DLBCL (Cerchietti et al. have shown efficacy in the treatment of DLBCL 2010).

1.1.2.3.3 HDAC inhibition

Histone deacetylase (HDAC) inhibitors have also been shown to repress BCL- 6 expression, as well as possessing apoptotic and cell cycle regulatory effects through other targets including repression of cMYC transcription and modulation of expression of BCL-2 family proteins (Bolden et al. 2006; Newbold et al. 2013a; 2013b; Bolden et al. 2013; Thompson et al. 2013). The preclinical promise of HDAC inhibition in aggressive B-cell lymphoma models has led to clinical use of vorinostat in relapsed and refractory DLBCL in which occasional complete and partial responses have been observed (Crump et al. 2008).

1.1.2.3.4 BET-bromodomain inhibition

Bromodomain and extra-terminal domain (BET) family members are epigenetic reader proteins that facilitate recruitment of transcriptional super-elongation complexes (SEC) following binding to acetylated lysine residues on histones (Dawson et al. 2012). DLBCL has been shown to have super-enhancer dependencies with approximately one-third of all of the BET family member protein, BRD4, localizing to fewer than two percent of occupied (Delmore et al. 2011; Chapuy of MYC targets, has led to rational targeting of DLBCL with BET-inhibitors such et al. 2013). This identification of super-enhancers in DLBCL, with co-activation as JQ1 and GSK525762. While the full mechanism of activity of BET inhibitors in aggressive lymphoma is currently under investigation in our laboratory, the preclinical activity of GSK525762 has led to clinical development in the treatment of a range of haematological malignancies including DLBCL and Burkitt lymphoma (NCT01943851).

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1.1.2.3.5 BCL-2 inhibition

With the development of increasingly selective BCL-2 inhibitors from navitoclax to venetoclax, and the unprecedented activity of these agents in indolent lymphoproliferative disorders such as CLL and mantle cell lymphoma, it is hardly surprising that venetoclax monotherapy was recently assessed in a cohort of relapsed and refractory B-cell lymphomas, including DLBCL (Roberts et al. 2016). However, venetoclax monotherapy was associated with reasonably poor overall response rates in DLBCL, suggesting that BCL-2 is not an oncogenic dependency to the majority of DLBCL cases (Roberts, Andrew. ‘Targeting BCL2: Tribulations and trials during clinical translation.’ New Directions in Leukaemia Research 2016, Noosa, Australia, 17 March 2016, Oration). Despite this, the empiric addition of BCL-2 antagonism with venetoclax to standard immunotherapeutic approaches for DLBCL is currently the subject of clinical investigation (NCT02055820). It would be expected that DLBCL with overexpression of BCL-2, such as double-hit lymphoma or transformed follicular lymphoma would be enriched for response to BCL-2 inhibition. BCL-2 and its targeting will be discussed in greater detail later in this chapter (sections 1.4.1.2.1 and 1.4.3).

1.1.2.3.6 RNA polymerase I inhibition

Elegant preclinical studies have shown a critical role for cMYC in binding ribosomal DNA and activating RNA polymerase I (Pol I) transcription of ribosomal RNA genes (Arabi et al. 2005; Grandori et al. 2005). The frequent dysregulation of MYC in aggressive lymphoma has led to interrogation of Pol I targeting as an approach in vivo in murine models of MYC driven lymphoma (Poortinga et al. 2011; Devlin to indirectly target the oncogenic activity of MYC, and this has shown efficacy et al. 2016). These studies have led to current clinical development of the Pol I inhibitor, CX-5461, in the treatment of a range of haematological diseases including aggressive lymphoma (ACTRN12613001061729).

1.1.2.3.7 Immunological approaches

1.1.2.3.7.1 PD-1 / PD-L1 Checkpoint inhibition

Avoidance of immune destruction is a relatively recent advancement in our understanding of fundamental principles of cancer cell survival (Hanahan and Weinberg 2011). Critical to this process is the B7-CD28 pathway which regulates activation and tolerance of T-cells (Sharpe and Freeman 2002). Through expression

10 Chapter 1 of programmed death ligands, PD-L1 and PD-L2, many different types of cancer cells are able to engage programmed death 1 (PD-1) to confer T-cell tolerance and enable avoidance of immune-mediated cancer cell destruction.

Relevance to haematological malignancy has been demonstrated through studies of Hodgkin lymphoma, whereby genetic studies of primary Hodgkin Reed-Sternberg including the locus for CD274 (encoding PD-L1) (Green et al. 2010). Indeed, cells has demonstrated a subset with amplification of chromosome 9p24.1,

(Roemer et more recent data suggests that amplification of PD-L1 is ubiquitous for Hodgkin al. 2016). Immune avoidance correlates with the typical histological appearance of lymphoma, involving amplification, copy number gain and polysomy Hodgkin lymphoma, whereby the malignant cells are often few in number within reactive cells including T-lymphocytes (Swerdlow et al. 2008). biopsy specimens and are surrounded by a reactive collar of inflammatory and

PD-1 blockade with agents such as nivolumab and pembrolizumab has provided a therapeutic approach to overcoming PD-1 / PD-L-mediated T-cell tolerance. Clinical trials of PD-1 blockade in solid organ malignancies and Hodgkin lymphoma has provided clear evidence of its value, though the role of PD-1 blockade in aggressive B-cell lymphoma remains the subject of a number of clinical trials that are currently in progress (Larkin et al. 2015; Brahmer et al. 2015; Le et al. 2015; Ansell et al. 2015).

1.1.2.3.7.2 Type II CD20 monoclonal antibodies

The successful use of the CD20 monoclonal antibody, rituximab, has unquestionably in the past century and has associated with approximately 25% more patients been the most significant advancement in the management of aggressive lymphoma achieving durable disease-free remissions than chemotherapeutic approaches without rituximab . When compared with type I CD20 monoclonal antibodies, type II CD20 monoclonal antibodies such as obinutuzumab (Coiffier et al. 2002) have been associated with reduced complement-dependent cytotoxicity and increased direct cell death and antibody-dependent cellular cytotoxicity (Mossner et al. 2010). The pivotal CLL11 clinical trial showed superiority of obinutuzumab, when compared to rituximab, in combination with chlorambucil for management of untreated CLL in older patients with comorbidities (Goede et al. 2014; 2015). of DLBCL NCT02596971, NCT02611323). Current clinical trials are assessing whether this benefit also extends to treatment

11 Chapter 1

1.1.2.3.7.3 CD38 monoclonal antibodies

CD38 is a cell surface glycoprotein located on B- and T-lymphocytes, plasma cells and natural killer cells, and its expression as part of the germinal centre reaction has been demonstrated on DLBCL cells (Alizadeh et al. 2000; Lokhorst et al. 2015). Targeting of CD38 with the monoclonal antibody, daratumumab, has led to dramatic responses in heavily pretreated patients with multiple myeloma, providing rationale for its clinical development in DLBCL (Lokhorst et al. 2015). However, whereas myeloma therapy has previously lacked a bona fide monoclonal antibody, use of rituximab or other CD20-monoclonal antibodies has been standard of care for DLBCL for almost 15 years . Refractory disease does not typically associate with downregulation of CD20 expression, indicating disease (Coiffier et al. 2002) persistence despite ongoing expression of the target. As daratumumab utilizes the same modalities of cell death as CD20-monoclonal antibodies (antibody- dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody- dependent phagocytosis and direct cell death / apoptosis) (Mossner et al. 2010), it is uncertain whether invoking the same mechanisms through a different cell surface antibodies as part of prior therapy. A phase II clinical trial of daratumumab in target will provide further benefit in patients previously exposed to monoclonal relapsed / refractory DLBCL, mantle cell lymphoma and follicular lymphoma is currently underway (NCT02413489) and should provide evidence of its utility in patients previously treated with CD20 monoclonal antibodies.

1.1.2.3.7.4 Radioconjugates

Radioconjugates represent a class of compounds that combine monoclonal antibodies with radioisotopes in order to deliver ionizing radiation locally to the tumour site (Rizzieri 2016). Hence, they may utilize existing monoclonal antibodies such as rituximab, though confer cytotoxicity through a different mechanism. Zevalin is a radioconjugate of rituximab and 90Yttrium, and its use in DLBCL has remained a topic of conjecture for some time (Rizzieri 2016). Despite clear activity with its upfront use in follicular lymphoma and in refractory follicular lymphoma and DLBCL, it has not been compared in randomization to existing salvage immunochemotherapy regimens (Rizzieri 2016). Due to this and limitations of its accessibility, radioconjugates remain a non-standard approach to DLBCL therapy.

12 Chapter 1

1.1.2.3.7.5 Bi-specific T-cell engagers

to engage cognate antigens on both target cells and T-cells in order to facilitate Bi-specific T-cell engagers (BiTE) are a class of monoclonal antibodies engineered T-cell mediated cytotoxicity (Mølhøj et al. 2007). Blinatumomab is a BiTE designed to engage CD19 (B-cells) and CD3 (T-cells) and is already approved by the Food and Drug Administration (FDA) for treatment of acute lymphoblastic leukaemia (Hoffman and Gore 2014). Importantly, a phase II clinical trial of blinatumomab for treatment of relapsed and refractory DLBCL has demonstrated a 43% overall response rate, which is supportive of its use in this disease group (Viardot et al. 2016).

1.1.2.3.7.6 CAR-T cells

Historical responses of refractory DLBCL treated with allogeneic bone marrow transplantation have provided evidence for activity of immune-modulation in this area, however, the transient nature of response and toxicity of such approaches precludes its use (Lazarus et al. 2010). Chimeric antigen receptor T-cells (CART) autologous T-cell use reducing the risk of graft versus host disease (Kochenderfer provide a mechanism for invoking this same immune response with the benefit of et al. 2015) due to the intensive labor required for bespoke production, recent use of CART . Although currently associated with significant cost of manufacturing therapy targeting CD19 or CD20 for relapsed and refractory DLBCL has been associated with dramatic responses, including complete and durable remissions (Kochenderfer et al. 2015; Wang et al. 2014). However, this approach is currently limited by cost, accessibility and the potential for tumour adaption through mutation of target leading to its reduced expression, as has been observed with CD19 in up to 15% of paediatric acute lymphoblastic leukaemias treated with this therapy (Sotillo et al. 2015).

1.1.2.4 Summary of current novel approaches

While the aforementioned therapeutic approaches provide a snapshot of current clinical trials of rational targeted therapies in the management of aggressive B-cell lymphoma, it remains to be seen whether any will provide a clear and durable

Of those listed above, only BET-, Pol I-, and HDAC-inhibition are rationally posited benefit for the majority of patients in the primary or relapsed and refractory setting. to oppose the oncogenic activity of MYC. Given the central role of dysregulated MYC activity in this group of diseases, direct targeting of MYC-directed transcription

13 Chapter 1 presents an attractive therapeutic approach. Very recent evidence suggests this may also be achieved through cyclin-dependent kinase (CDK)-9 inhibition of RNA polymerase II (Pol II) activation as described later in section 1.3.5.5.

1.1.3 Murine modelling of MYC-driven B-cell lymphoma

1.1.3.1 The Eµ-Myc model

The Eµ-Myc lymphoma, resulting from insertion of the Igh-cMyc transgene from a model was first described in 1985 as a faithful model of Burkitt spontaneously arising murine plasmacytoma (Adams et al. 1985). Resultant transgenic mice develop aggressive B-cell lymphoma / leukaemia with a relatively short latency of between two-to-six months. However, unlike Burkitt lymphoma, which has a germinal centre B-cell immunophenotype, only approximately one- third of spontaneously arising Eµ-Myc lymphomas match this phenotype, with the remainder immunophenotypically consistent with precursor B-cell leukaemia / lymphoma.

Furthermore, Eµ-Myc mice typically display a transient polyclonal increase in B-cells

B-cell outgrowth which coincides with additional mutational events (Sidman et al. in leukaemic phase, prior to regression of lymphocytosis, then a final monoclonal 1993; Eischen et al. 1999; Klapproth and Wirth 2010). Hence, despite frequently being referred to as a faithful model of Burkitt lymphoma, Eµ-Myc actually represents a relatively heterogeneous lymphoma model with variable precursor / germinal centre B-cell phenotype corresponding to inconstant maturation of the postulated normal counterpart cell prior to malignant transformation. Therefore, Eµ-Myc may more correctly be representative as a model of MYC-dysregulated immature B-cell lymphoma, rather than Burkitt lymphoma per se. This notion is further supported by sequencing data from our group and collaborators in which the mutational landscape of Eµ-Myc manuscript revision under review). These studies have highlighted similarities lymphoma has been defined (Lefebure et al, with Burkitt lymphoma such as mutations of p53 and ADP-ribosylation factor (ARF1), as well as differences such as lack of mutation of Myc itself and high frequency mutation of the proposed tumour suppressor, BCL6 corepressor (BCOR).

Use of the Eµ-Myc lymphoma model has been published extensively by our laboratory and others (Adams et al. 1985; Whitecross et al. 2009; Mason et al. 2008; Shortt et al. 2013; Devlin et al. 2016), as it possesses the valuable property of being serially transplantable into cohorts of syngeneic recipient mice with

14 Chapter 1 virtually 100% uniform engraftment. This allows for accurate assessment of using a relevant model for toxicity examination. Furthermore, by utilising a efficacy of therapeutic interventions and for the presence of a therapeutic window syngeneic immunocompetent model, immunological mechanisms of activity and resistance are intact and not negated as they would be in immunocompromised models required for xenograft studies.

1.1.3.2 Other spontaneous murine models of MYC-driven B-cell lymphoma

A number of other murine models somewhat analogous to Eµ-Myc lymphoma have been derived and are considered equivalent in their pathogenesis and utility. These include generation of iMycEµ mice through insertion of mus musculus cMyc cDNA into the Igh locus upstream of the Eµ enhancer (Han et al. 2010), His and a similar model using His6-tagged cMyc, termed Myc (Park et al. 2005). Furthermore, other elegant models have been developed to demonstrate the molecular dependencies of MYC-driven lymphoma through methods such as Cre- recombination of constitutively activated PI3K together with MYC (Sander et al. 2012), but serial transplantability of these lymphomas for therapeutic testing has not yet been reported.

1.1.3.3 Xenografting human MYC-driven B-cell lymphoma

Xenografting of human MYC-driven B-cell lymphoma cell lines into immunocompromised mice such as the non-obese diabetic severe combined γnull) strain has historically provided the only mechanism by which to interrogate lymphoma biology and response to therapeutics immunodeficient (NOD-SCID IL2R in vivo. Recently, successful passaging of primary patient-derived xenografts directly from biopsy through to establishment of serially-transplantable tumours has been reported and will provide an invaluable resource for further genetic and therapeutic studies (Chapuy et al. 2016). However, this resource is not currently available to the wider research community for therapeutic testing.

1.2 MYC

1.2.1 MYC forms, structure and function

v-myc gene within oncogenic retroviruses causing myelocytomatosis, a fulminant form of leukaemia MYC was first discovered through identification of the and sarcoma affecting chicken (Sheiness and Bishop 1979). The different MYC

15 Chapter 1 family members are encoded by related genes, LMYC, NMYC, cMYC and SMYC (Adhikary and Eilers 2005). MYC protein contains a basic helix-loop-helix leucine zipper (bHLH/LZ) domain, through which the LZ domain confers binding to its heterodimerization partner, MAX, while the bHLH domain provides a binding platform for DNA (Murre et al. 1989). Together with MAX, they bind conserved E-box DNA sequences (5’-CACGTG-3’) allowing for their transcriptional regulation of target genes (Amati et al. 1992).

MYC activity has been associated with multiple cellular processes, although due to its established role in tumorigenesis, it is most commonly described in the context of cell growth and proliferation (Dang 2012). While MYC binds to more than 25,000 sites in the , only approximately 15% of all encoded genes are under direct transcriptional regulation by MYC (Adhikary and Eilers 2005).

1.2.2 Regulation of MYC

MYC acts downstream of several receptor pathways that activate or repress MYC activity. Due to the numerous roles that MYC plays in cell growth and proliferation and potential for malignant transformation of tissues, it is under stringent regulatory control at the levels of transcription, translation

Transcriptional control of MYC has been associated with a number of transcription and post-translational modification (Adhikary and Eilers 2005; Longo et al. 2015). factors, and a number of other DNA structures have also been shown to regulate MYC such as G-quadruplexes (Dang 2012; Hurley et al. 2006). At the level of translation, MYC is further regulated through mechanisms such as micro-RNA (miR) repression of translation mediated through conserved regions within the 3’-untranslated region (UTR) (Cannell et al. 2010).

with stabilisation conferred through phosphorylation at Ser62 by MAPK, whereas Furthermore, MYC is also subject to significant post-translational modification Thr58 phosphorylation by glycogen synthase kinase (GSK) predisposes to ubiquitination and degradation (Gregory and Hann 2000; Adhikary and Eilers 2005; Lutterbach and Hann 1994). These processes are critical to regulation of MYC activity, and explain its extremely short 15-20 minute half-life.

Cellular protection from acute overexpression of MYC is conferred by checkpoint activation leading to apoptosis. MYC itself upregulates expression of p53 and ARF1 (repressing MDM1-mediated inhibition of p53)(Figure 1.2) (Zindy et al. 1998; Eischen et al. 1999; Klapproth and Wirth 2010). Following its activation,

16 Chapter 1

ARF1

MDM2

MYC

p53

BCL-2 BIM

PUMA NOXA APOPTOSIS

Figure 1.2: MYC interactions with apoptosis pathways

MYC upregulates expression of p53 and ARF1, which in turn inhibits MDM2 to remove its repression of p53 activity. Activated p53 upregulates pro-apoptotic PUMA and NOXA leading to apoptosis induction. MYC also suppresses expression of anti-apoptotic BCL-2 and induces expression of pro-apoptotic BIM, further potentiating apoptosis.

Arrows denote direct upregulation or activation; bars denote direct inhibition.

17 Chapter 1 p53 then upregulates pro-apoptotic PUMA and NOXA, with subsequent activation of BAX and BAK leading to intrinsic apoptosis induction (Figure 1.2) (Michalak et al. 2008). Independent of this process, MYC has been shown to directly suppress anti-apoptotic BCL-2 expression and induce pro-apoptotic BIM expression (Figure 1.2), which further potentiates apoptosis conferred by p53 activation, while also contributing to p53-independent apoptosis induction (Eischen et al. 2001; Hoffman and Liebermann 2008; Hemann et al. 2005). NOXA has been shown to directly bind and degrade myeloid cell leukaemia 1 (MCL-1), but is balanced by stabilisation of MCL-1 conferred by BIM binding (Czabotar et al. 2007). Furthermore, apoptosis of malignant cells leads to transforming growth factor (TGF)-β secretion from macrophages, providing a signal to promote senescence (Reimann et al. 2010).

1.2.3 Dysregulated MYC activity in cancer

Following the seminal discovery of cMYC

translocation as the defining lesion MYC of Burkitt lymphoma, studies across a broad range of tumours have identified (Dalla-Favera et al. 1982; Schmitz et al. 2012; Beroukhim et al. 2010). However, frequent translocation, amplification and gain of function mutations of dysregulation of MYC induction of apoptosis and senescence that occurs following excessive MYC activity alone is insufficient to effect tumorigenesis, largely due to (Klapproth and Wirth 2010). Elegant studies of murine Myc-driven lymphoma mutations that occur and synergise with dysregulated MYC activity in order to and then human Burkitt lymphoma have identified a number of recurrent somatic promote neoplastic cell growth, such as p53 and ARF (section 1.1.3.1), and ID3 and TCF3 (section 1.1.2.2) (Eischen et al. 1999; Schmitz et al. 2012; Richter et al. 2012).

1.2.4 MYC and positive transcription elongation factor B

promotes its numerous cellular functions. Notably, co-immunoprecipitation A significant body of evidence exists to explain the mechanisms by which MYC studies have demonstrated MYC to directly bind and recruit cyclin T1, the binding partner of CDK9 (Kanazawa et al. 2003). Together, cyclin T1/CDK9 form the positive transcription elongation factor, P-TEFb, which is the key activator of transcriptional elongation (Marshall et al. 1996). Further evidence of this critical interaction was demonstrated by chromatin immunoprecipitation (ChIP), whereby MYC and P-TEFb were demonstrated to co-occupy E-boxes (MYC target sites) in a process that could be abrogated by pharmacological inhibition of P-TEFb with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) (Gargano et al. 2007).

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Beyond recruitment of P-TEFb to its target sites, MYC has also been shown to regulate P-TEFb activity through regulation of CDK9 expression. Notably, the slow proliferative rate of Myc-/- expression of Myc mutant forms lacking the DNA binding domain, but possessing fibroblasts can be almost completely rescued by the transactivation domain, via MYC-dependent upregulation of CDK9 mRNA translation (Cowling and Cole 2007). Hence, MYC is directly involved in regulating P-TEFb, which is the critical determinant of Pol II pause-release into productive elongation discussed below.

1.2.5 RNA polymerase II activation and termination

Transcriptional initiation, pausing, elongation and termination are critical molecular processes regulating Pol II activity that are required for appropriate gene expression (Smith and Shilatifard 2013). Pol II is recruited by general transcription factors (TFIIB, TFIID, TFIIE, TFIIF, TFIIH) to the promoter-proximal region downstream of the transcription start site, resulting in pre-initiation complex formation (Jonkers and Lis 2015; Sainsbury et al. 2015). Of the general transcription factors, TFIIH confers structure-altering properties including ATPase and helicase activity to assist unwinding of DNA through negative superhelical tension in order to produce a conformation favorable for transcription (Kim et al. 2000b). Following initiation and entry, Pol II is rapidly paused by its negative regulators, negative elongation factor (NELF) and DRB sensitivity-inducing factor (DSIF), which itself comprises the heterodimer of Spt4 and Spt5 (Figure 1.3a) (Missra and Gilmour 2010; Sansó and Fisher 2014).

Initiation and pausing of Pol II requires activity of CDK7, a component of TFIIH. CDK7 phosphorylates Pol II subunit RPB1 at Ser5 and Ser7 residues of heptapeptide repeats within the carboxy-terminal domain (CTD), contributing to release of TFIIE and recruitment of DSIF with resultant pausing (Figure 1.3a) (Glover-Cutter et al. 2009; Larochelle et al. 2012). Furthermore, CDK7 has been shown to directly activate CDK9 through phosphorylation of its threonine-rich loop (T-loop) (Larochelle et al. 2012).

Following its activation, CDK9 performs a triad of phosphorylation to release Pol II pausing into productive transcriptional elongation (Figure 1.3b). This triad consists of phosphorylation of the Pol II RPB1 CTD Ser2 residues of the heptapeptide repeats, phosphorylation of NELF leading to its dissociation, and phosphorylation of Spt5 of DSIF to convert it to a positive elongation factor (Marshall and Price 1992; 1995; Marshall et al. 1996; Larochelle et al. 2012; Sansó and Fisher 2014;

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Figure 1.3: CDK7 and CDK9 regulation of Pol II pause-release and productive elongation

(a) CDK7 phosphorylates the RNA Polymerase II carboxy-terminal domain (CTD) at Ser5 and Ser7 residues, leading to a conformational change favorable for recruitment of Pol II inhibitors, negative elongation factor (NELF) and DRB- sensitivity-inducing factor (DSIF) with resultant Pol II pausing. CDK7 also phosphorylates CDK9 to induce its activation. (b) Activated CDK9 phosphorylates: NELF leading to its dissociation; DSIF to convert it to positive elongation factor (yellow); and the RNA polymerase II CTD at Ser2 residues leading to transcriptional elongation.

Block arrows denote direct phosphorylation; dashed arrow denotes indirect recruitment; black bar denotes repression; S denotes serine residue of the CTD; P denotes phosphorylation.

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a

CDK9

CDK7 NELF DSIF

YSPTSPS/YSPTSPSCTD RNA polymerase II

Paused

b

CDK9

NELF

DSIF YSPTSPS/YSPTSPSCTD RNA polymerase II

Elongation

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Jonkers and Lis 2015). Productive transcriptional elongation ensues.

In metazoans, termination of transcription of coding mRNA occurs via binding of stimulatory factor (CstF) and cleavage factor (CF)-I and –II, all of which cleavage and polyadenylation specificity factor (CPSF) to the body of Pol II, and bind the Pol II CTD in its Ser2 phosphorylated state (Porrua and Libri 2015). Furthermore, both CPSF and CstF recognize and bind the polyadenylation site site. Senatexin binds this site and possibly contributes to a conformational change within the 3’-UTR of nascent RNA, conferring specificity with regard to the cleavage allowing the exoribonuclease, XRN2, to bind the free 5’ end, degrade the cleavage product and terminate transcription (West et al. 2004; Porrua and Libri 2015). This process is commonly referred to as the ‘torpedo model’ of transcription termination. Recent evidence has demonstrated XRN2 to be a substrate of P-TEFb phosphorylation, whereby inhibition of CDK9 or mutation of XRN2 (Thr 439) leads to ineffective transcription termination (Sansó et al. 2016).

Hence, despite several processes controlling activation and termination of Pol II transcription, the above description outlines a sequential and non-random series of activations governed by CDKs (CDK7 activating CDK9; CDK9 activating and removing inhibition of Pol II; and CDK9 activating XRN2) to ensure orderly regulation of Pol II activity.

1.3 Cyclin-dependent kinases

The cyclin-dependent kinases represent a conserved group of protein kinases involved in cell cycle regulation and transcriptional control. As their name suggests, CDKs are inactive until bound by one of their cognate cyclin binding activation (Hochegger et al. 2008; Malumbres 2014). This process separates the partners, typically followed by phosphorylation of the T-loop as a final step of C-helix and activation domain, which are otherwise preventing substrate access to the catalytic pocket (Malumbres 2014). In terms of subcellular localisation, inactive CDKs are predominantly cytoplasmic and once activated translocate to the nucleus to perform their kinase activity (Pines and Hunter 1991; Napolitano boxes’, the site of interaction with cyclins comprising amino acid residues stacked et al. 2002). Structurally, CDKs are defined by the presence of one or more ‘cyclin α-helices (Malumbres 2014). into five Early descriptions of CDKs included CDK1-6, predominantly involved in cell cycle regulation as described below (section 1.3.2, Figure 1.4) (Malumbres et al. 2009).

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CDK7 Cyclin H

CDK4/6

CDK2

Retinoblastoma G1 S CDK2 G0 M G2

CDK1 CDK1 Cyclin A

Figure 1.4: Interaction of CDKs with the cell cycle

CDK7 activates other CDKs through phosphorylation. CDK4/6 phosphorylation of retinoblastoma removes its repression of transcription factors associated with cell cycle entry (G0/G1). CDK2, CDK1 and their cognate cyclins are involved in G1/S, S/G2, and G2/M transitions. P denotes phosphorylation; G0, gap 0/resting phase; G1, gap/growth 1 phase; S, DNA synthesis phase; G2, gap/growth 2 phase; M, mitosis. Black arrows denote activation; black bar denotes repression.

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cycle regulation, but rather regulation of transcription (Malumbres et al. 2009). Next, description of CDK7-10 identified CDKs with predominant roles not in cell More recent elucidation of the roles and structures of other protein kinases and very recent expansion up to CDK20 (Malumbres et al. 2009). Furthermore, a their dependencies on cyclins has led to their reclassification as CDK11-13, then group of separate protein kinases with similarity to CDKs, but without evidence of cyclin binding partners, has been termed the CDK-like (CDKL) family and comprises CDKL1-5 (Malumbres et al. 2009). For the purpose of this review, a brief description of each CDK with regards to its function, regulation and role in cancer is provided below.

1.3.1 Transcriptional cyclin-dependent kinases

The ‘transcriptional CDKs’ were a later addition to the CDK family, and were largely the presence of a ‘cyclin box’ and their requirement for activation through cyclin reclassified as CDKs due to their similarity to classical CDKs 1-6 with regards to heterodimerization (Malumbres et al. 2009; Malumbres 2014). This group comprises CDK7, 8, 9, 10, 11, 12, 13, 19 and 20, all of which have associations with transcriptional processes rather than distinct cell cycle regulatory roles (Figure 1.5).

1.3.1.1 CDK7

CDK7 and the yeast ortholog Kin28 are components of the TFIIH transcription factor and perform several critical cellular processes in conjunction with its activating partner cyclin H, including phosphorylation of the Pol II CTD (Ser5 and Ser7) described above (in 1.2.5) to regulate transcription initiation and pausing. Furthermore, CDK7 functions as a CDK-activating kinase (CAK) in that it phosphorylates a number of CDKs in their T-loop to complete their activation (Figure 1.4) (Larochelle et al. 2012). This latter process directly implicates CDK7 into a myriad of processes involving independent CDKs, both transcriptional and non-transcriptional.

Importantly, genetic studies have revealed absence or mutation of Cdk7 to mediate embryonic lethality not due to defective transcription (Pol II CTD Ser5 phosphorylation is normal, indicating redundancy in this role), but rather due to loss of CAK function limiting Cdk1 and Cdk2 activity (Ganuza et al. 2012). Furthermore, conditional loss of Cdk7 in adult tissues was associated with premature aging due to loss of progenitor cells. Tissues with low proliferative indices, however, remained

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Transcriptional CDKs

CDK7 Group III Cyclins CDK8 (Cyclin C Group) CDK9 CDK10 CDK11 CDK12 CDK13 CDK19 CDK20

Non-Transcriptional CDKs

CDK1 Group I Cyclins CDK2 (Cyclin B Group) CDK3

CDK4 CDK6

CDK5 Group II Cyclins CDK14 (Cyclin Y Group) CDK15 CDK16 CDK17 CDK18

Figure 1.5: Subgrouping of CDKs and their cognate cyclin binding partners

Transcriptional CDKs are activated by binding the group III cyclins (cyclin C group). Non-transcriptional CDKs comprise two main subfamilies; the ‘cell cycle’ associated CDKs are activated by group I cyclins (cyclin B group), whereas the CDK5 family are activated by binding the group II cyclins (cyclin Y group).

25 Chapter 1 phenotypically normal. Consistent with highly proliferative cells being sensitive to loss of CDK7 activity, a selective inhibitor of CDK7 (achieved through binding a cysteine residue remote from the kinase domain and blocking its activity) is cell lines and provides a rationale for further testing of CDK7 inhibitors in cancer associated with significant activity against T-cell acute lymphoblastic leukaemia (Kwiatkowski et al. 2014).

1.3.1.2 CDK8

CDK8 and its yeast ortholog Srb10 are components of the kinase module of Mediator, a large multi-unit complex directly and indirectly involved in regulation of Pol II-mediated transcription (Conaway and Conaway 2011; Larivière et al. 2012). Importantly, Mediator can only bind Pol II in the absence of the kinase module, as the addition of this module occludes the site of Pol II binding (Knuesel et al. 2009). For this reason, CDK8 has been associated with a predominant repressive role on transcription. Furthermore, even when unbound from Pol II, CDK8-cyclin C is associated with phosphorylation of a number of transcription factors and initiation factors, negatively regulating their activity and repressing transcription in a process distinct from its blocking of Pol II interaction (Conaway and Conaway 2011). Included in this group of CDK8 substrates is cyclin H, whereby phosphorylation impairs activity of CDK7 (Egly and Coin 2011). Hence CDK8 indirectly regulates CDK7 transcriptional activity.

CDK8 is critical for normal development, as Cdk8 knockout is embryonic lethal at the stage of implantation (Westerling et al. 2007). The discovery of frequent dysregulation of CDK8 or cyclin C in melanoma and colorectal carcinoma has led to interest in development of CDK8 inhibitors, though their use in vitro has only demonstrated slight repression of neoplastic cell line proliferation (Xu and Ji 2011; Koehler et al. 2016).

1.3.1.3 CDK9

Elucidation of the function of P-TEFb comprising CDK9 and cyclin T has been fundamental to our current understanding of regulation of transcription elongation (Marshall and Price 1992; 1995; Marshall et al. 1996; Jonkers and Lis 2015). As described above (section 1.2.5), P-TEFb substrates of Pol II CTD (Ser2), NELF and DSIF are all associated with Pol II pause-release into productive elongation (Marshall et al. 1996; Larochelle et al. 2012; Sansó and Fisher 2014; Jonkers and Lis 2015). Furthermore, the recent description of CDK9 regulating activation of

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XRN2 has provided further evidence for the sequential and controlled processes of transcription elongation and termination (Sansó et al. 2016). CDK9 has also been shown to directly phosphorylate p53 at multiple serine residues, though the effect of this on p53 function is uncertain (Radhakrishnan and Gartel 2006).

As opposed to the oscillating expression of cell cycle-related cyclins regulating CDK activity, transcriptional CDK-associated cyclins are generally expressed at more constant levels (Malumbres 2014). Hence, regulation of CDK9 through dimerization with cyclin T and other post-translational modifications is required. the CAK properties of CDK7 (Larochelle et al. 2012) and acetylation at lysine 44 Such activating modifications of CDK9 include phosphorylation of the T-loop by within the kinase domain(Fu et al. 2007). Furthermore, acetylation of cyclin T1 leads to dissociation from HEXIM1 and 7SK snRNA, relieving their repression of P-TEFb activity (Cho et al. 2009).

In contrast, ubiquitination of HEXIM1 by HDM2 strengthens its interaction with P-TEFb and results in greater inhibition of its activity (Lau et al. 2014). Negative regulation of CDK9 activity can also be conferred by the combination of acetylation at lysine 44 and lysine 48 by GCN5 and P/CAF (Sabo et al. 2008). Finally, it has been reported that cyclin T1 interacts with SCFSKP2 directly to target CDK9 for proteasomal ubiquitination and degradation (Kiernan et al. 2001), however, contrasting evidence suggests that CDK9 is not subject to direct SCFSKP2–mediated regulation (Garriga et al. 2003). CDK9 itself is a stable protein with a reported half-life of four to seven hours, though when overexpressed it is rapidly degraded within one hour due to lack of stabilisation (Garriga et al. 2003).

bona fide knockout mouse models are available. It is generally accepted that the reason Genetic interrogation of CDK9 has historically been difficult and no for this is early embryonic lethality, a hypothesis supported by inhibition of Mus musculus 2011), and death of Drosophila melanogaster embryogenesis with the pan-CDK inhibitor, flavopiridol (Oqani et al. knockdown of Cdk9 (Eissenberg et al. 2006). Furthermore, while knockout of flies at metamorphosis following cyclin T1 has proven elusive, CycT2-/- mice are embryonic lethal, supporting the hypothesis of P-TEFb as being a critical requirement for normal embryogenesis (Kohoutek et al. 2009).

During the preparation of this thesis, publication of elegant RNA interference (RNAi) studies of murine Myc-driven hepatocellular carcinoma models demonstrated a dependence upon Cdk9 (Huang et al. 2014). Mechanistically, genetic silencing of

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Myc or Cdk9 resulted in similar reduction of Pol II occupancy in the gene body of NPM1 as an illustrative example. Moreover, MYC expression of human hepatocellular carcinoma cell lines was shown to be predictive of response to pharmacologic antagonism of CDK9 with a novel inhibitor, and in vivo assessment of the inhibitor against representative cell lines xenografted into immunocompromised mice was a potential dependency of MYC-driven cancers upon CDK9 and provide further associated with significant clinical responses. These experiments highlighted rationale for the studies described in this thesis.

1.3.1.4 CDK10

Elucidation of the function of CDK10 has arisen from understanding the developmental abnormality, STAR syndrome, characterised by renal and anogenital malformations, telecanthus and toe syndactyly (Malumbres 2014; Guen et al. 2013). STAR syndrome is associated with mutations in FAM58A (encoding cyclin M), impairing the binding of CDK10–cyclin M. The CDK10-cyclin M heterodimer is indirectly involved in transcription as it phosphorylates the transcription factor, ETS2, marking it for proteasomal degradation (Guen et al. 2013). Hence impaired CDK10-cyclin M activity leads to accumulation of ETS2, allowing for increased ETS2-mediated transcription of RAF with resultant upregulation of the MAPK pathway (Guen et al. 2013). Importantly, extension of these studies demonstrated the upregulation of MAPK upon CDK10 knockdown to be associated with tamoxifen resistance of breast cancer cell lines (Guen et al. 2013). This implicates a tumour suppressive role for CDK10 and opposes any rationale for targeting CDK10 in cancer therapy.

1.3.1.5 CDK11

CDK11 proteins are encoded by the two highly related CDK11A and CDK11B genes. of lower molecular weight (Malumbres et al. 2009). Hence, isoforms of CDK11 Furthermore, translational modifications lead to further truncated proteins include CDK11Ap110, CDK11Bp110, CDK11Ap58 and CDK11Bp58. The CDK11-cyclin L complex regulates L-Mediator by controlling association of the kinase complex and the S-Mediator complex (Drogat et al. 2012). L-Mediator directly co-activates Pol II activity by providing a bridge between the Pol II CTD and transcription factors, and it has been recently shown that inactivating mutations of Cdk11 in yeast confer similar changes to global gene expression as mutations affecting the kinase module of Mediator containing Cdk8 (Drogat et al. 2012).

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Notably, CDK11 has recently been implicated as a critical oncogenic requirement for breast carcinoma growth and angiogenesis, osteosarcoma cell survival and intrinsic multiple myeloma cell survival (Chi et al. 2015; Tiedemann et al. 2012; Duan et al. 2012; Feng et al. 2014). Triple-negative breast cancer cells demonstrate high CDK11 nuclear expression with immunohistochemistry and targeting of CDK11 with RNAi is associated with loss of viability of these cells (Kren et al. 2015) the exact mechanism by which this is occurring remains unclear. However, genetic . These reports suggest CDK11 to play significant oncogenic roles, though

G2-M transition and required for mitotic spindle formation (Petretti et al. 2006). interrogation of HeLa cells has shown specific expression of CDK11p58 at the Furthermore, loss of Cdk11 activity in embryogenesis is associated with similar mitotic defects (Malumbres 2014). Hence, CDK11 appears to play critical roles in normal transcriptional processes and its dysregulation in cancer is recurrently approach. described. These findings propose CDK11 targeting as a potential novel therapeutic

1.3.1.6 CDK12

CDK12 and CDK13 are highly similar proteins orthologous to yeast Ctk1 (Malumbres 2014; Malumbres et al. 2009; Bartkowiak et al. 2010). RNAi studies of Drosophila melanogaster, yeast and human cancer cell lines have demonstrated CDK12-cyclin K to have Pol II CTD Ser2 phosphokinase activity (Bartkowiak et al. 2010; Liang et al. 2015; Kohoutek and Blazek 2012). Importantly, P-TEFb performs the majority of global Pol II Ser2 phosphorylation, whereas CDK12 has a propensity for phosphorylation of Pol II Ser2 toward the middle and 3’ end of genes (Bartkowiak et al. 2010).

when it was demonstrated by RNA-sequencing that cells depleted of CDK12 show Further specificity for CDK12 regulation of transcriptional processes was observed lower expression of DNA damage response genes and RNA processing factors, leading to defects in RNA processing (Liang et al. 2015; Greifenberg et al. 2016). Hence, despite the suggestion of redundancy in Pol II CTD Ser2 phosphorylation (involving CDK9, CD12 and CDK13, described next), the alterations in Ser2 phosphorylation observed upon genetic silencing of CDK9 or CDK12 suggests that this redundancy is incomplete and therefore therapeutic targeting of individual CDKs may perturb their Pol II regulatory function.

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1.3.1.7 CDK13

Due to the similarities to CDK12 from an evolutionary and structural perspective, it is not surprising that CDK13 exhibits Pol II CTD phosphorylation properties. Indeed, CDK13 has been demonstrated to phosphorylate the CTD at both Ser2 and Ser5, with a propensity for Pol II already phosphorylated at Ser7 (Greifenberg et al. 2016). Supportive of a disparate role that CDK13 plays from CDK12 and CDK9, genetic depletion of CDK13 in the HCT116 colorectal cancer cell line demonstrated enrichment for CDK13 activity in genes associated with growth signalling pathways, in contrast to the DNA damage response genes associated with CDK12 depletion (Greifenberg et al. 2016).

CDK13 has recently been proposed as a novel oncogene due to the discovery of frequent CDK13 (Edwards et al. 1998; Kim et al. 2012) gene amplification in hepatocellular carcinoma been reported. . However, no specific therapies targeting CDK13 have yet

1.3.1.8 CDK19

CDK19 is most closely related to CDK8 from an evolutionary and functional perspective (Malumbres et al. 2009; Malumbres 2014). CDK19 is associated with the kinase module of Mediator, though not in the presence of CDK8. Hence, CDK19 plays a similar role to CDK8 in providing the enzymatic kinase properties to the kinase module when not bound to Pol II. It is hypothesized that the promiscuity differing substrates (Sato et al. 2004). of the kinase module (regarding CDK8 or CDK19) confers specificity of binding to

1.3.1.9 CDK20

From an evolutionary perspective, CDK20 (cell-cycle related kinase) most closely resembles CDK7 and also binds cyclin H (Malumbres et al. 2009). However, CDK20 is not associated with Pol II CTD phosphorylation, nor does it possess CAK properties (Malumbres 2014). CDK20 has been shown to activate intestinal cell kinase through phosphorylation of Thr157, and this process has been implicated in the pathogenesis of both glioblastoma and hepatic carcinoma (Fu et al. 2006; Yang et al. 2013; Feng et al. 2011). Mechanistically this was shown to associate with activation of β-catenin / T cell factor signalling advancing cell cycle progression potential targeting for cancer therapy. (Feng et al. 2011). These findings implicate an oncogenic role for CDK20 and its

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1.3.2 Non-transcriptional cyclin-dependent kinases

Much of our understanding of CDK function and regulation arose from initial descriptions of CDK activity pertaining to cell cycle regulation. It is now considered that the so-called ‘cell cycle’ CDKs fall within three subfamilies; CDK1, CDK4 and CDK5 (Figure 1.5) (Malumbres et al. 2009; Malumbres 2014). These CDKs share common properties including conserved sequences in the glycine-rich

(Malumbres 2014). loop (G-loop), post-translational modification of which regulates their activity

In general, phosphorylation of Thr14 and / or Thr15 by and Myt1 leads to inhibition of CDK function, with the exception being that Thr15 phosphorylation of CDK5 is activating. Members of the family use their phosphatase properties to remove the inhibitory phosphorylation of these residues in order to improve substrate binding (Boutros et al. 2007). CDKs are also subject to negative regulation through inhibition by members of the INK4 and Cip/Kip families, such as p21Cip1, p27Kip1 and p57Kip2, all of which interfere with the CDK/cyclin heterodimerization (Malumbres 2014). Hence, control of CDK activity occurs predominantly through binding of cyclins, the expression of which oscillates through the cell cycle, and

2014). post-translational modification of CDKs limiting their activation (Malumbres

1.3.2.1 CDK1

CDK1 is the human ortholog of yeast Cdc28. CDKs from the CDK1 subfamily (CDK1- 3) show promiscuity in their binding, dimerizing with cyclins A, B, E and perhaps C (Malumbres 2014). Cell cycle progression was previously considered to require the activity of CDK1 at mitosis, as well as the CDKs (2, 3, 4 and 6). However, genetic studies have revealed that in the absence of all other interphase CDKs, CDK1 can effectively bind interphase cyclins leading to phosphorylation of retinoblastoma (Rb) and release of transcription factors to drive expression of target genes and effect cell cycle progression (Santamaría et al. 2007). Indeed, this level of redundancy is exclusive to CDK1, as its deletion is associated with redundancy exists for CDK1 itself (Santamaría et al. 2007). early embryonic lethality (approximately day 2.5), indicating that insufficient

In terms of canonical function, expression of cyclins A and B during G2 lead to activation of CDK1, which then co-ordinates mitotic processes such as maturation and separation of centrosomes, condensation of and entry into

31 Chapter 1 mitosis (Figure 1.4) (Malumbres 2014). CDK1 also regulates cell cycle progression through phospho-activation of the -promoting complex ubiquitin , leading to proteasomal degradation of other cell cycle related proteins (Fujimitsu et al. 2016). Finally, it was recently demonstrated that the transcriptional oscillation of components of the cell cycle machinery is co-ordinated around CDK1 activity (Banyai et al. 2016). Hence, CDK1 provides the critical platform on which all cell cycle progression is based.

Having such critical roles in cell cycle progression, it would be predicted that dysregulated CDK1 activity may be oncogenic and therefore targeted for therapeutic purposes. Indeed, inhibition of CDK1 has been shown to induce apoptosis of MYC-dependent mouse lymphoma and hepatocellular carcinoma through downregulation of CDK1-regulated anti-apoptotic expression (Goga et al. 2007).

1.3.2.2 CDK2

Seminal studies targeting CDK2 demonstrated its primary role governing transition of the cell cycle from G1 to S-phase (Figure 1.4) (Pagano et al. 1993). However, the original descriptions of a critical dependency upon CDK2 involved use of kinase-dead mutant forms which retained the ability to dimerize with cyclins. While these observations remain valid today, the absolute dependence on CDK2 for this transition has since been questioned by the redundancy conferred in absence of CDK2 (Santamaría et al. 2007). The principle reason for these by CDK1, even though the efficiency of progression at this checkpoint is reduced discordant observations upon cell cycle progression in the absence of CDK2 is that kinase dead CDK2 was diluting the available pool of cyclins for CDK1 activation (Pagano et al. 1993; Santamaría et al. 2007). Hence, while CDK2 is still preferred for facilitating the G1/S-transition, its presence is not critical in order for this that Cdk2-/- mice are viable, albeit smaller in size and infertile owing to reduced checkpoint to be passed. Supportive of this redundancy process is the finding development of reproductive tissues (Berthet et al. 2003).

1.3.2.3 CDK3

CDK3 is most closely related to CDK2 in terms of structure and function Cdk3-/- mice show no phenotypic

(Malumbres et al. 2009). However, the finding that role of CDK3 (Ye et al. 2001). Despite this, CDK3-mediated phosphorylation of abnormality and maintain normal fertility are evidence for a less significant

32 Chapter 1 activating transcription factor 1 (ATF1) at Ser63 has been shown to contribute to maintenance of glioblastoma cell lines, and RNAi depleting Cdk3 reduces ATF1 function and is detrimental to target cell proliferation (Zheng et al. 2008).

1.3.2.4 CDK4

Phosphorylation of Rb by CDK4-cyclin D or CDK6-cyclin D is considered the initiating event driving cells into the cell cycle (Figure 1.4) (Kato et al. 1993; Dowdy et al. 1993). Upon phosphorylation of Rb, E2F transcription factors are released and bind dimerization partner (DP) proteins, leading to E2F-DP-driven expression of genes governing transition into S-phase. Due to the potential for uncontrolled cell growth and division following this activity, CDK4/6 are subject to greater inhibitory regulation than most CDKs. Aside from Cip/Kip family- mediated repression, CDK4/6 are also repressed by the INK4 family comprising p16INK4a, p15INK4b, p18INK4c and p19INK4d (Gil and Peters 2006).

translocation involving CCND1 (encoding cyclin D) and the IgH promoter region as An oncogenic role for CDK4/6-cyclin D was established by the finding of a recurrent event driving mantle cell lymphoma (Swerdlow et al. 2008). Similarly, loss of tumour suppression conferred by deletion or mutation of the CDKN2A locus (encoding INK4a/ARF) or deletion of Rb, all support the critical role of this axis in many cancers (Weinberg 1991; Gil and Peters 2006). This knowledge has set the scene for the current interest in clinical development of CDK4/6-inhibitors discussed later (section 1.3.5.4).

1.3.2.5 CDK5

As CDK5 lacks any direct effect on the classical model of the cell cycle, it was previously rather unique in its presence within the original six CDKs. However,

(CDK5, 14, 15, 16, 17 and 18) now comprises the largest subfamily (Malumbres as the reclassification of other proteins as CDKs has occurred, the CDK5 family et al. 2009). Genetic studies have demonstrated CDK5 to play a predominant role and perinatal death of Cdk5-/- mice and its association with neurodegenerative in neuronal development, as shown by the significant neurological abnormalities diseases such as Alzheimer’s disease (Ohshima et al. 1996; Patrick et al. 1999). More recently, an unbiased screen of RNAi hairpins demonstrated targeting of CDK5 to synergise with proteasomal inhibition for cytotoxicity against a number development of CDK5 inhibitors for treatment of myeloma (Kumar et al. 2014). of myeloma cells (Zhu et al. 2011). These findings have led to interest in the clinical

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1.3.2.6 CDK6

As for CDK4, CDK6-cyclin D is associated with phosphorylation of Rb leading to entry into the cell cycle (Figure 1.4). Notably, haematopoietic tissues have been shown to express relatively higher levels of CDK6 than CDK4, indicating CDK6 as was supported by the discovery that therapeutic targeting of CDK6, but not CDK4, a potential greater dependency in these models (Placke et al. 2014). This finding can release the myeloid differentiation block of mixed-lineage leukaemia (MLL)- translocated acute myeloid leukaemia (AML) (Placke et al. 2014). Furthermore, studies of BCR-ABL translocated B-acute lymphoblastic leukaemia have provided key information regarding internal feedback loops suppressing CDK6. CDK6, but not CDK4, is associated with a transcription complex that increases transcription of p16INK4a, and upon mutation or deletion of p16INK4a, uncontrolled cell growth and proliferation occurs due to dysregulated CDK6 activity (Kollmann et al. 2013). Furthermore, this complex upregulates VEGF-A expression, conferring angiogenic activity that is independent of CDK6’s kinase function. A critical requirement for CDK6 in haematopoietic lineages is supported by mouse modelling, whereby Cdk6-/- mice are viable though exhibit defects in red blood cell development, whereas mice without both Cdk4 and Cdk6 are associated with late embryonic lethality and phenotypically show reduced global haematopoiesis (Tsutsui et al. 1999).

1.3.2.7 CDK14

Characterisation of CDK14 (PFTK1) has demonstrated it to associate with and p21Cip1, and to be involved with cell cycle progression in an Rb-dependent manner (Shu et al. 2007). CDK14 has been implicated in the maintenance of several different solid cancer subtypes, and its knockdown has been shown to reduce tumour proliferation and invasion (Pang et al. 2007; Yang et al. 2015; Zheng et al. 2015). Importantly, the PFTK1 genetic locus on chromosome 7q21-22 has been demonstrated to be amplified in a number of cancer types including cases stage of disease at presentation and inferior patient outcomes (Pang et al. 2007). of sporadic Burkitt lymphoma, and this amplification is associated with advanced

1.3.2.8 CDK15

Very little published literature exists to delineate the functions of CDK15. From an evolutionary perspective, CDK15 is within the CDK5 subfamily of CDKs, and the only described function of CDK15 is in phospho-stabilisation of survivin (Thr 34),

34 Chapter 1 conferring resistance to TRAIL-induced apoptotic stimuli in colorectal and breast cancer cell lines (Malumbres et al. 2009; Park et al. 2014).

1.3.2.9 CDK16

CDK16 (PCTAIRE1) represents another member of the CDK5 subfamily, is ubiquitously expressed and predominantly cytoplasmic in localisation (Charrasse et al. 1999). CDKs 16-18 are unique in that they share a common PCTAIRE sequence in the C-terminus, where amino- and carboxy-terminal domains abut the catalytic domain (Malumbres 2014). Regulation of activation is achieved via phosphorylation of the amino-terminal domain, preventing binding of cyclin Y to the catalytic domain. Notably, CDK16 has been demonstrated to interact with p35 in the neuromuscular junction and is subject to phospho-regulation by CDK5, indicating that these proteins are not only related from an evolutionary perspective but also functionally (Cheng et al. 2002).

CDK16 is highly expressed in melanoma, breast, prostate and cervical cancer cell lines. Its depletion is associated with reduced phosphorylation of p27 (Ser10) leading to accumulation of p27 and a reduction in proliferation (Yanagi et al. 2014a; 2014b). Phenotypic abnormalities resulting from genetic silencing of CDK16 could be fully rescued by concomitant depletion of p27 (Yanagi et al. 2014a). Finally, CDK16 depletion is associated with TRAIL-induced apoptosis of multiple solid cancer cell lines, concomitant with caspase 8 activation and degradation of RIPK1 (Yanagi et al. 2015). Hence, targeting of CDK16 presents an attractive therapeutic option for cancer, though to date the only described CDK16 inhibitor (SNS-032) actually inhibits several CDKs at nanomolar concentrations and therefore its et al. 2011). efficacy cannot be attributed specifically to CDK16 targeting in isolation (Walsby

1.3.2.10 CDK17

CDK17 (PCTK2 / PCTAIRE-2) also clusters within the CDK5 subfamily from an evolutionary perspective (Malumbres 2014). From the scant literature available regarding its structure and function, it is highly conserved and along with CDK18 and P-histone H4 has been implicated in amyloid precursor protein development in Alzheimer’s pathogenesis (Chaput et al. 2016).

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1.3.2.11 CDK18

Aside from the aforementioned role in amyloid precursor protein deposition, CDK18 has been demonstrated to be involved with DNA damage regulation and depletion of CDK18 has been associated with cytostasis at S-phase concomitant genome stability (Chaput et al. 2016; Barone et al. 2016). Specifically, genetic with replication stress defects, and these phenotypic abnormalities could be fully rescued by exogenous CDK18 re-expression (Barone et al. 2016). Through the regulation of replication stress response, it would be expected that CDK18 would represent a putative oncogene, however, the only association of CDK18 with lymphoma cells whereby its depletion was only associated with a minimal increase cancer has been through boutique selective RNAi modification of cutaneous T-cell in proportion of non-viable cells (Şahin et al. 2014). 1.3.3 Cyclins

α-helices known as the cyclin box (Malumbres 2014). The remainder The cyclins comprise approximately 29 proteins and are defined by the presence of of the protein consists of an amino-terminal domain for interacting with the CDKs five stacked and a carboxy-terminal domain which is required for the folded protein structure. The family is broadly divided into three subgroups with related functions. The group I cyclins (cyclin B group) interact with cell cycle-related CDKs, the group II cyclins (cyclin Y group) interact with the CDK5 family, and the group III cyclins (cyclin C group) interact with the transcriptional CDKs (Figure 1.5) (Malumbres 2014). As with the CDKs, aberrant cyclin expression has been well-described as an initiating event in lymphoma with the notable example of translocation t(11;14) (q13;q32) leading to cyclin D dysregulation driving mantle cell lymphomagenesis (Swerdlow et al. 2008; Campo and Rule 2015).

1.3.4 Cyclin-dependent kinase-like family

Five additional proteins are related to the CDK family and possess kinase activity, though with the distinction that they lack the requirement for activating cyclin heterodimerization (Malumbres et al. 2009). These are the CDK-like (CDKL) family and comprise CDKL1-5. Despite their evolutionary relation to the CDKs, little more is known regarding their function aside from genetic studies of Cdkl2-/- mice displaying impaired cognition (Gomi et al. 2010). No reports of their dysregulation in cancer have been published to date.

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1.3.5 Cyclin-dependent kinase inhibitors

With such critical roles in cell proliferation and transcriptional regulation, CDK inhibition for therapeutic gain has been pursued across a spectrum of haematological and solid organ malignancies. Early generation ‘pan-CDK to mechanistically interrogate their activity and also provided a signal as to inhibitors’ such as flavopiridol and seliciclib provided the tools through which due to disappointing results in treatment of patients with CLL (Byrd et al. 2005; therapeutic benefit. However, their clinical development was largely discontinued 2007; Lin et al. 2009). Relevant literature pertaining to key CDK inhibitors is outlined below, especially regarding their use in lymphoid malignancies. The IC50 values for these inhibitors are outlined in Table 1.1.

1.3.5.1 Flavopiridol

Flavopiridol (alvocidib, L868275, HMR-1275) is derived from Dysoxylum binacteriferum and is the most studied historical pan-CDK inhibitor (Blachly et al. 2016). Since early studies describing its ability to reduce phosphorylation to inhibit P-TEFb and transcriptionally repress expression of key molecular of CDK1 in breast cancer cell lines, a number of papers have defined its ability determinants of cell survival such as BCL-2 in CLL and MCL-1 in multiple myeloma (Worland et al. 1993; Chao and Price 2001; Konig et al. 1997; Gojo et which to produce more selective CDK inhibitors in order to demonstrate which al. 2002). Furthermore, derivatives of flavopiridol have provided the means by targets are critical for its activity in different cellular contexts (Kim et al. 2000a). Despite early promise through preclinical studies, disappointing results arising from clinical trials including few complete responses, toxicity including cytokine release syndrome, and unfavourable pharmacokinetics requiring impractical dose

2009; Byrd et al. 2005; 2007). However, recent evidence suggests its combination schedules, largely led to discontinuation of flavopiridol use in the clinic (Lin et al. investigation of its utility in this disease group (Zeidner et al. 2015). with chemotherapy may add benefit for patients with AML, leading to renewed

1.3.5.2 Seliciclib

Seliciclib (roscovitine, CYC202) has also been studied extensively in preclinical models of haematologic malignancy. These include demonstration of reduction in MCL-1 and expression in multiple myeloma and mantle cell lymphoma cell lines, reduced phosphorylation of Pol II in CLL and DLBCL cell lines and primary

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IC50 [nM]

Dinaciclib Flavopiridol Seliciclib A1592668.1 Palbociclib AZ-CDK9

CDK1 3 30 330 2000 >10000 24 CDK2 1 170 220 2000 >10000 254 CDK3 360 CDK4 100 >10000 50 11 3252 CDK5 1 170 270 >9800 >10000 8005 CDK6 80 530 16 3868 CDK7 800 >9800 767 CDK8 >10000 CDK9 4 20 230 93 <3 CDK10 Clinical Phase II Approved Phase II Preclinical Approved Preclinical Development

Table 1.1: Inhibitory profiles of CDK inhibitors

Published and unpublished in vitro IC50 values for CDK inhibition by the various CDK inhibitors, and the current stage of clinical development reached.

38 Chapter 1 patient samples, and the associated phenotypic changes that occur including G2 cell cycle arrest and apoptosis induction (Raje et al. 2005; MacCallum et al. 2005; preclinical studies of seliciclib have demonstrated interference with other critical Lacrima et al. 2007; Rogalińska et al. 2009; Lacrima et al. 2005). Furthermore, cellular processes including DNA damage repair, as evidenced by abrogation of expression and associated non-homologous end-joining responses to DNA damaging cytotoxic agents (Federico et al. 2010). Despite the development of more selective CDK inhibitors, seliciclib is still being assessed in clinical trials with the contemporary example of its combination with sapacitabine for patients with advanced solid tumours (NCT00999401).

1.3.5.3 Dinaciclib

Unlike the pan-CDK inhibitors, development of dinaciclib (SCH727965, Merck, against CDKs 1, 2, 5 and 9 (Paruch et al. 2010; Parry et al. 2010). Early preclinical Boston, MA, USA) heralded a CDK inhibitor with more specific nanomolar activity studies demonstrated its ability to reduce phosphorylation of Pol II, associate with apoptosis induction through biomarkers of cell viability and cleavage of PARP, and cause a G2 cytostasis (Parry et al. 2010). Extension of these studies has shown dinaciclib to induce apoptosis of osteosarcoma and CLL cells, and have in vivo activity against xenografted pancreatic carcinoma and melanoma (Fu et al. 2011; Johnson et al. 2012a; Feldmann et al. 2011; Abdullah et al. 2011).

Aside from the cytostatic and transcriptional effects of dinaciclib, this compound has also been demonstrated to show activity through CDK1/5-dependent inhibition of the unfolded protein response with reduction in XBP-1 in myeloma cell lines in vitro and xenografts in vivo (Nguyen and Grant 2014). Furthermore, despite the ability of dinaciclib to inhibit the ATP-binding kinase domain of target CDKs, it has also been demonstrated to possess protein-protein bromodomain inhibitory targets and epigenetic mechanisms (Martin et al. 2013). properties, making it a unique tool with potential efficacy through both direct with advanced solid malignancies, clinical development of dinaciclib rapidly Following demonstration of its tolerability in first-in-human studies of patients progressed to phase I/II studies in CLL, acute leukaemia and multiple myeloma (Nemunaitis et al. 2013; Gojo et al. 2013; Kumar et al. 2014; Flynn et al. 2015). However, clinical development of dinaciclib for CLL was abruptly halted due to perceived market competition following the marked clinical responses observed in clinical trials of venetoclax and ibrutinib separately (Byrd et al. 2013; Roberts

39 Chapter 1 et al. 2015).

1.3.5.4 Palbociclib

Palbociclib (PD-0332991) is the most clinically advanced CDK inhibitor currently in use. Palbociclib inhibits CDK4/6 at low nanomolar concentrations, and does not target any other CDK at sub-micromolar concentration (Liu and Gray 2015). Following demonstration of its ability to potently suppress phosphorylation of Rb in colorectal cancer and melanoma cell lines, it has been used across a range of solid organ malignancies leading to seminal publication of its activity in the treatment of patients with breast carcinoma (Fry et al. 2004; Yoshida et al. 2016; Turner et al. 2015). These latter results led to accelerated approval of palbociclib by the FDA in 2015.

1.3.5.5 Other cyclin-dependent kinase inhibitors

A number of further CDK inhibitors have recently emerged through their use in associated with reduction in phosphorylation of Pol II and CDK7, as well as reduced published preclinical studies. SNS-032 has a broad inhibitory profile and has been expression of MCL-1, BCL-2, X-linked inhibitor of apoptosis (XIAP), CDK9 and CDK2 in AML and breast carcinoma cell lines (Walsby et al. 2011; Xie et al. 2014). RO-3306 is a CDK1 inhibitor with potency against AML cell lines effected through upregulation of pro-apoptotic BAX expression (Kojima et al. 2009). SU9516 is reported to be a selective CDK2 inhibitor, though the effects on Pol II and MCL-1 through which the authors propose it exerts its apoptotic activity would suggest that it likely also has activity against transcriptional CDKs (Gao et al. 2006). However, apoptosis induction has also been demonstrated through inhibition of CDK1/2 with purvanolol in MYC-expressing cells in a survivin-dependent manner (Goga et al. 2007).

compounds have recently been reported. Targeting of a remote cysteine residue In terms of specific targeting of transcriptional CDKs, a number of preclinical

CDK7, and this compound was notably observed to have activity against T-acute outside of the kinase domain has enabled development of a specific inhibitor of lymphoblastic leukaemia cell lines through downregulation of RUNX1 expression (Kwiatkowski et al. 2014). RGB-286638 is reported to be an inhibitor of transcriptional CDKs and demonstrates reduction in Pol II phosphorylation with reduction in MCL-1 and XIAP, though an associated reduction in phosphorylation of Rb suggests concomitant inhibition of CDK4 or CDK6 (Cirstea et al. 2013).

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Finally, a number of novel CDK9 inhibitors were recently reported including i-CDK9, LDC000067, PC585 (AZ’5576, AZCDK9) and PHA-767491 (Lu et al. 2015; Albert et al. 2014; Garcia-Cuellar et al. 2014; Huang et al. 2014). While these compounds are generally associated with reduction in phosphorylation of Pol II and global transcriptional repression, a few notable features of these compounds are reported. LDC000067 treatment of AML cell lines is associated with a reduction in MYC expression, whereas prolonged exposure of HeLa cells to i-CDK9 is associated with a BRD4-dependent upregulation of MYC expression

MLL-translocated AML models, and is a synonym for AZCDK9 that is described in (Albert et al. 2014; Lu et al. 2015). PC585 demonstrated significant activity against studies in this thesis. Finally, despite the activity of PHA-767491 against MYC- driven hepatocellular carcinoma being attributed to CDK9 inhibition, another through reducing phosphorylation of Cdc7 target sites in replicative DNA (Huang group has published studies demonstrating its efficacy at inhibiting DNA synthesis et al. 2014; Montagnoli et al. 2008). Despite these inconsistencies, the majority of publications describing inhibition of transcriptional CDKs invariably link at least part of their activity to induction of apoptosis.

1.4 Apoptosis

Apoptosis is the process of programmed cell death, initiated by a diverse range of extracellular and intracellular processes that ultimately leads to cell destruction. It hallmark of cancer (Beroukhim et al. 2010; Hanahan and Weinberg 2000). is a critical function of tissues, and its dysregulation is perhaps the most significant Apoptosis is initiated by two distinct processes mediated by differing pathways, with each culminating in the activation of intracellular serine proteases termed caspases, which are the executioners that mediate cell death (Zou et al. 1997). The two pathways leading to apoptosis induction are the ‘intrinsic’, ‘mitochondrial’ or ‘stress’ pathway (Figure 1.6) and the ‘extrinsic’ or ‘death receptor’ pathway (Adams and Cory 2007; Czabotar et al. 2014).

1.4.1 Intrinsic apoptosis

The intrinsic pathway consists of three distinct families of proteins that tightly regulate cell fate. The Bcl-2 homology domain 3 (BH3)-containing ‘BH3-only’ pro-apoptotic proteins (BIM, BID, BIK, BMF, PUMA, NOXA, HRK) utilise their

BH3 domain to bind the anti-apoptotic proteins (BCL-2, MCL-1, BCL-XL, BCL-W, A1, BCL-B) in order remove the latters’ regulation of effector proteins BAK, BAX or BOK (Figure 1.6) (Adams and Cory 2007; Czabotar et al. 2014; Sattler et al.

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Cytokine deprivation p53 activation Cellular stressors

BID BIM PUMA NOXA

BMF BIK HRK BAD

BAK BAK BAK BAX A1 BCL-XL' MCL-1 BCL-W BAX BAX

BCL-B BCL-2

BAK BAK APAF-1 BAX BAX Caspase 9

Cytochrome c

Figure 1.6: Intrinsic apoptosis pathway

displace bound effector proteins (grey diamonds) from anti-apoptotic proteins Following various stimuli (upper figure), BH3-only proteins (blue diamonds) (yellow ovals). Freed effector proteins then change conformation and homo- dimerise to activate (red diamonds) and perforate the mitochondrial outer membrane. This leads to release of cytochrome c, which in turn activates caspase 9 on a scaffold of APAF-1 on the mitochondrial outer membrane. Activated caspase 9 then further propagates the downstream apoptosis signalling through activation of other caspases (not shown).

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1997; Willis et al. 2007). Historical ‘direct’ and ‘indirect’ models of BAX/BAK activation were described, in which the fundamental difference was whether the BH3-only proteins also directly engage and activate BAX/BAK. For some time, this evidence of BH3-only proteins co-immunoprecipitating with effector proteins had direct activation model was not favoured, largely due to the fact that no definite been shown and that apoptosis could be induced without this BH3-only/effector interaction (Adams and Cory 2007; Willis et al. 2007). However, this rationale did not recognise the transient nature of such an interaction in order to induce conformational changes in BAX/BAK that contribute to their full activation (Dai et al. 2011; Westphal et al. 2013). Description of the crystal structures of BAX and BH3-only proteins have provided a comprehensive understanding of these two groups of proteins, and led to the current accepted doctrine that regulation of effector protein activation results from both direct and indirect activation conferred by dynamic interactions between the three groups of proteins (Czabotar et al. 2013; 2014).

A diverse range of stressors have been shown to regulate activation of the intrinsic pathway, including cytokine deprivation, expression of anti-apoptotic and pro- apoptotic proteins and intracellular damage (Chittenden et al. 1995; Vaux et al. 1988). Following activation of this pathway, BAK, BAX (and less commonly BOK) mediate mitochondrial damage causing release of cytochrome c, ultimately leading to caspase-9 activation through apoptotic protease-activating factor 1 (APAF-1) on the mitochondrial surface (Zou et al. 1997; Cecconi et al. 1998; Jürgensmeier et al. 1998).

1.4.1.1 Effector proteins

BAX, BAK and BOK are very similar in appearance to their anti-apoptotic counterparts and share the BH1, BH2, BH3 and BH4 domains (Figure 1.7) (Czabotar et al. 2013; 2014). Interaction of the BH1, BH2 and BH3 domains of anti-apoptotic proteins forms a hydrophobic groove within which the BH3 domain of the effector proteins is bound (Czabotar et al. 2014). Upon their release from anti-apoptotic protein repression, they homo-oligomerize and create pores on the mitochondrial outer membrane (MOM) (Westphal et al. 2013; O’Neill et al. 2016). This mitochondrial damage is a critical step to apoptosis induction and caspase activation. Seminal studies have confirmed the downstream position of the effector the presence of BH3-only proteins (Zong et al. 2001; Wei et al. 2001). proteins, as absence of both BAK and BAX significantly impedes apoptosis, even in

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BH3-only proteins BH3 TM BIM, PUMA, NOXA, tBID, BIK, HRK, BAD, BMF

Anti-apoptotic proteins BH4 BH3 BH1 BH2 TM

BCL-2, MCL-1, BCL-XL, BCL-W, A1

Effector proteins BH4 BH3 BH1 BH2 TM BAK, BAX, BOK

Figure 1.7: Protein structure of BCL-2 family members

Structures of the BH3-only, anti-apoptotic and effector proteins are shown. The BCL-2 homology (BH) domains are denoted by blue boxes and the transmembrane domains (TM) are denoted by red boxes.

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1.4.1.1.1 BAK

BAK (BCL-2 homologous antagonist / killer) is encoded by the BAK1 gene located on chromosome 6. The encoded protein contains four BH domains (BH1-4) cloned, elegant studies demonstrated overexpression of BAK to stimulate apoptosis rendering similarity to the other effector and anti-apoptotic proteins. When first

BCL-2 (Chittenden et al. 1995). BAK is constantly tethered to the MOM through of fibroblasts and partially abrogate protection conferred from overexpression of the α9 helix at its hydrophobic C-terminus and it is here that it is repressed by anti-apoptotic protein binding, or upon release, spontaneously activates and forms homo-oligomers that perforate the MOM (Dai et al. 2011; O’Neill et al. 2016; Westphal et al. 2013).

BAK has been shown to predominantly interact directly with only a select group of anti-apoptotic proteins (BCL-XL, MCL-1 and A1) (Willis et al. 2005; Adams and Cory 2007). It has recently been shown that mutation of murine Bak(Glu75) / human

BAK(Q77) removes Bcl-xL repression of Bak through disrupted binding without

L in repression of Bak activity (Lee et al. 2016). Despite an established role in apoptosis and tissue impairing apoptosis induction, confirming the critical role of Bcl-x regulation, Bak-/- mice appear phenotypically normal, maintain normal fertility and are notable only for thrombocytosis in the peripheral blood which is refractory to thrombocytopenia-induction by the BCL-2/-XL inhibitor, ABT-737 (Lindsten et al.

L binding and the evidence for BCL-X as the critical determinant of platelet lifespan (Mason 2000; Mason et al. 2007). LThese findings are predicted by BAK / BCL-X fertility has been observed with Bax-/- mice, the generation of Bak-/- / Bax-/- mice et al. 2007). Whereas lineage specific defects in lymphoid development and suggesting a level of redundancy between Bak and Bax function (Knudson et al. was associated with significant perinatal death and developmental abnormalities, 1995; Lindsten et al. 2000). This redundancy would also explain why deletion or mutation of BAK is not described recurrently in any form of cancer or autoimmune disease in the literature (Beroukhim et al. 2010).

1.4.1.1.2 BAX

BAX (BCL-2-associated X protein, also known as Bcl-2-like protein 4) is encoded by the BAX gene on chromosome 19. It also comprises the BH1-4 domains, conferring similarity to BAK and anti-apoptotic proteins. As opposed to the membrane- bound location of BAK, BAX is predominantly cytosolic until a conformational change induced upon activation leads to exposure of its α9 helix and membrane

45 Chapter 1 localisation (Westphal et al. 2013). Nuclear magnetic resonance spectroscopy has demonstrated an activation site distinct from the anti-apoptotic , and elucidation of the crystal structure of BAX has revealed the dynamic conformational changes following its activation (Gavathiotis et al. 2008; Czabotar et al. 2013). BAX is considered more promiscuous than BAK, and likely binds all of the anti-apoptotic proteins (Willis et al. 2007). The proposed redundancy between BAK and BAX likely explains the mild phenotypic changes observed in Bax-/- mice and the fact that recurrent mutation in BAX is not a frequent occurrence across a landscape of different cancer types, although recurrent mutation in a particular subtype of colorectal cancer has been described (Rampino et al. 1997).

1.4.1.1.3 BOK

BOK (BCL-2 related ovarian killer) is named for its predominant expression in ovarian and female reproductive tissues, though it is actually expressed widely at lower levels (Ke et al. 2012). Due to the phenotypic effects of deletion of both BAK and BAX or dependent on the activity of BAK / BAX to mediate its apoptotic effects. This from many cellular systems, BOK had been considered less significant Box-/- mice are phenotypically normal, maintain normal fertility and are not predisposed to increased cancer risk (Ke et al. hypothesis was reinforced by the finding that 2012). However, recent studies have shown that BOK alone can induce apoptosis in the absence of both BAK and BAX in several different cell types (Einsele-Scholz et al. 2016). Furthermore, reconstitution of murine haematopoiesis with foetal liver haematopoietic cells transduced for knockdown of Box, Bak and Bax together potentiates the lymphocytosis observed when compared to the Bak and Bax to be a bona fide combinatory knockdown (Ke et al. 2015). Together, these studies confirm BOK effector protein, though the significant body of evidence to date apoptosis induction and dysregulation in cancer. Despite this, BOK has been shown would suggest that BOK is less significant than its effector protein counterparts to to be recurrently mutated across a number of different cancer types (Beroukhim et al. 2010).

1.4.1.2 Anti-apoptotic proteins

from delineation of the BCL2 gene at the breakpoint region of the translocation The first insights into the genetic and molecular processes of apoptosis dawned translocated to come under the regulation of the IGH promoter region (Tsujimoto defining human follicular non-Hodgkin lymphoma, whereupon this gene is et al. 1984). Since that time, the structure, canonical and non-canonical functions

46 Chapter 1 of each of these group of ‘BCL-2 family’ members has been described. These proteins contain the BH1, BH2, BH3 and BH4 domains (Figure 1.7) and also the hydrophobic α9 helix through which they interact with the MOM (Czabotar et al. 2014).

1.4.1.2.1 BCL-2

Following initial description of the BCL2 gene on chromosome 18 from studies of follicular lymphoma, seminal mechanistic studies were performed in which retroviral insertion of the Bcl2 gene into haematopoietic cells from wild type and Eµ-Myc mice demonstrated co-operation with cMyc to cause B-cell lymphoma (Vaux et al. 1988). Furthermore, overexpression of Bcl-2 was found to confer survival to cytokine-dependent haematopoietic cell lines upon withdrawal of cytokines. These experiments provided the basis from which our current understanding of the anti-apoptotic function of BCL-2 family members is derived. BCL-2 is widely expressed, though appears to play key roles in renal tissues, melanocytes and Bcl-2-/- mice, whereby absence of Bcl-2 is associated with absence of mature lymphocytes, mature lymphoid cells. These findings were made upon observation of premature grey coat and reduced survival owing to development of renal failure due to polycystic kidneys (Veis et al. 1993). The phenotypic effects of Bcl-2 loss were demonstrated to be through its canonical function when further genetic lesions were introduced, demonstrating that even a single allelic loss of Bim was Bcl-2-/- phenotype (Bouillet et al. 2001). While BCL-2 sufficient to rescue the associated with NOXA or BAK when compared to its associations with other BH3- displays significant promiscuity in terms of binding partners, it is less commonly only proteins or BAX (Czabotar et al. 2014).

Aside from the canonical anti-apoptotic role of BCL-2, non-canonical roles regulating cell cycle progression and tumour suppression have also been shown (Huang et al. 1997; La Coste et al. 1999; Vairo et al. 2000). Notably, BCL-2 positively regulates expression of cell cycle inhibitors p27 and p130, leading to cytostasis seminal works overexpressing Bcl-2, whereby retroviral insertion of Bcl2 into the with a G1 arrest (Vairo et al. 2000). These findings support those of the original cytokine-dependent haematopoietic cell lines was protective against apoptosis, though with an associated cytostasis preventing proliferation (Vaux et al. 1988).

Through the initial description of BCL-2 from studies of follicular lymphoma, an oncogenic role was immediately established. Furthering this, high BCL-2 expression or presence of a translocation involving BCL2 has been shown to confer

47 Chapter 1 poor prognosis to DLBCL (Johnson et al. 2012b; Hu et al. 2013; Savage et al. 2016).

BCL-2 discussed later in this chapter. These findings highlighted the need for development of the putative inhibitors of

1.4.1.2.2 MCL-1

MCL-1 is the protein encoded by the MCL1 gene on chromosome 1. Sequence isolation from the ML-1 AML cell line (Kozopas et al. 1993). As with all BCL-2 similarity to BCL-2 was shown when MCL-1 was first described following its family members, this protein contains the BH1-4 domains and the full-length form is tethered to the MOM through its α9 helix. MCL-1 displays both promiscuity and selectivity in terms of its binding partners. It binds both BAK and BAX, although it has been shown to interact with NOXA more frequently than with other BH3-only proteins (Willis et al. 2005; ADAMS and Cory 2007; Czabotar et al. 2014).

MCL-1 is predominantly associated with an anti-apoptotic canonical role through the typical BH3-only protein / effector protein interactions, however, a shorter splice variant termed ‘MCL-1S’ also exists and lacks the BH1 and BH2 domains and α9 helix (Bae et al. 2000). The resultant protein is in effect a ‘BH3-only’ protein and has been shown to interact with the longer ‘MCL-1L’ form directly. Overexpression of splicing factor 3B1 (which regulates the ratio of MCL-1L:MCL-1S) has been of MCL-1S has confirmed its pro-apoptotic function, and pharmacological inhibition shown to increase MCL-1S levels and potentiate apoptosis (Gao and Koide 2013). A further ‘extra-short’ splice variant termed ‘MCL-1ES’ has also been described and exhibits similar pro-apoptotic properties to MCL-1S (Kim et al. 2009).

Aside from the MCL-1 variants involved in apoptosis, a splice variant that localises within mitochrondria has been described through studies of mouse embryonic Mcl1 deletion to be associated with abnormal mitochondrial morphology, however, this was fibroblasts (MEFs). In these experiments, investigators observed not observed when only the full-length MCL-1 isoform was removed (Perciavalle et al. 2012). Interrogation of this system revealed a smaller splice variant to be residing within the mitochondrial inner membrane, and mutation of this variant anti-apoptotic function conferred by full-length MCL-1. significantly disrupted mitochondrial ATP production, though did not disrupt the

Similar to BCL-2, MCL-1 has also been demonstrated to play a role in cell cycle regulation, though through a distinct binding motif that other BCL-2 family members lack. MCL-1 interacts with proliferating cell nuclear antigen (PCNA),

48 Chapter 1 and overexpression of MCL-1 causes cytostasis in a PCNA-dependent manner that could be rescued through mutation of the PCNA interacting site on MCL-1 (Fujise isoforms that may augment anti-apoptotic effects through stabilisation of other et al. 2000). These findings demonstrate other non-canonical roles for MCL-1 cellular processes.

MCL-1 is subject to phospho-regulation involving distinct molecular pathways. AKT regulates expression of glycogen synthase kinase 3 (GSK3), and it has been shown that GSK3 phosphorylation of MCL-1(Ser155 and Ser159) leads to its ubiquitinylation and degradation, and this process could be rescued by enforced expression of AKT or pharmacological inhibition of GSK3 (Cross et al. 1995; Maurer et al. 2006). In opposition, phosphorylation of MCL-1(Thr92 and Thr163) by extracellular signal-related kinase (ERK)-1 and MCL-1(Ser121) by c-Jun N-terminal kinase (JNK)-1 have been shown to stabilise MCL-1 and prevent its degradation (Ding et al. 2008; Kodama et al. 2009). Importantly, this stabilisation of MCL-1 could be overcome through pharmacological inhibition of ERK phospho-activation with (Ding et al. 2008). The interplay of regulators of MCL-1 are critical as studies using an array of differing methodologies have demonstrated MCL-1 protein to have an extremely short half-life of less than 30 minutes (Nijhawan et al. 2003; Thomas et al. 2010; Perciavalle and Opferman 2013).

Relevant to this thesis, insertion of an MCL1 transgene into the haematolymphoid compartment in mice has led to develop B-cell lymphoma at high incidence (Zhou et al. 1998; 2001). Furthermore, previous studies have demonstrated accelerated lymphomagenesis upon silencing of tuberin in the Eµ-Myc model through upregulation of mTORC1 signalling (Mills et al. 2008). Pharmacological inhibition of resultant lymphomas with rapamycin was associated with rapid apoptosis induction through translational downregulation of MCL-1. These

Myc lymphoma. Transcriptional upregulation of MCL-1 has also been shown to be findings implicate mTORC1 as a translational regulator of MCL-1 expression in Eµ- related to other stimuli including cytokines such as interleukin-3 and members of the signal transducers and activators of transcription (STAT) family (Wang et al. 1999). Transcriptional repression of MCL-1 has previously been shown to associate with use of historical pan-CDK inhibitors in vitro, concomitant to a reduction in Pol II (Ser2) phosphorylation as a biomarker of CDK9 inhibition transcriptional regulation of MCL-1. (Gojo et al. 2002; MacCallum et al. 2005). This finding implicates CDK9 in the

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MCL-1 is widely expressed and is critical in embryonic development, as Mcl1-/- mice are embryonic lethal and cannot be rescued even with concomitant bi-allelic deletion of Bax (Rinkenberger et al. 2000). Mcl1 in cardiac myocytes also leads to apoptosis induction, precipitating heart Inducible tissue-specific deletion of failure (Wang et al. 2013c). Notably for the hypothesis generation of studies described in this thesis, Mcl1 deletion in haematopoietic cells leads to loss of the entire haematopoietic compartment, indicating a complete dependence of normal haematopoietic cells (Opferman et al. 2005). Finally, during the latter course of experimental work described herein, a series of reports using elegant genetic driven B-cell lymphoma upon MCL-1 (Kelly et al. 2014; Aubrey et al. 2015; Grabow techniques have been published that confirm the oncogenic dependency of MYC- et al. 2016a; 2016b). A critical oncogenic role has been predicted across a range

MCL1 in assessed tumours (Beroukhim et al. 2010). These of solid organ and haematological malignancies, based on the finding of frequent amplification of of MCL-1 function, and whether a potential therapeutic window may exist given findings highlight the need to investigate methods of direct or indirect antagonism the critical role that MCL-1 plays in a range of cellular processes across a broad array of tissue types.

1.4.1.2.3 BCL-XL

B-cell lymphoma-extra large (BCL-XL, also known as BCL-2-like 1 isoform 1) is the longer anti-apoptotic isoform of the BCL-2-like protein 1 (BCL-X) encoded by the BCL2L1 gene on chromosome 20. Similar to MCL-1, a shorter isoform termed BCL-

XS (B-cell lymphoma-extra short) has been described, and this variant interacts with BCL-XL and BCL-2 and is pro-apoptotic in function (Lindenboim et al. 2001).

BCL-XL is highly promiscuous, binding the majority of BH3-only proteins as well as BAK and BAX (Willis et al. 2005; 2007). BCL-X is critical to tissue development as BCL2L1-/- mice are embryonic lethal at day 13 owing to neuronal and hepatic haematopoietic cell apoptosis (Motoyama et al. 1995). Furthermore, extension of these studies showed BCL-X to be critical to survival of maturing lymphocytes expressing B220 +/- IgM, but not the mature B-lymphocyte compartment. Pharmacologic and genetic interrogation of BCL-X has proven its central role in controlling platelet lifespan, and the associated thrombocytopenia that arises from use of BCL-XL inhibitors (Mason et al. 2007). Despite this potential dose-limiting toxicity of BCL-XL BCL2L1 across a number of different human cancers inhibitors, a rationale for its targeting exists due to the finding (Beroukhim et al. 2010). of frequent amplification of

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1.4.1.2.4 A1

BCL-2-related protein A1 (A1) most closely resembles MCL-1 in that it associates with the majority of BH3-only proteins including a preference for NOXA rather than BAD, and it also binds both BAK and BAX (Willis et al. 2005; 2007; Simmons et al. 2008). A1 also appears to have a predominant effect in haematological cells, as short hairpin RNA (shRNA) targeting A1 is associated with a reduction in lymphocytes, and knockout of the A1a gene causes reduced mast cell and granulocyte survival (Hamasaki et al. 1998).

1.4.1.2.5 BCL-W

BCL-2-like protein 2 (BCL-W or BCL2L2) displays similar properties to BCL-2 in that it binds the majority of BH3-only proteins with a preference for BAD rather than NOXA, and it typically associates with BAX but not BAK (Chen et al. 2005a; Willis et al. 2005; 2007). BCL-W is not critical for normal development of most tissues, and it has been shown that Bcl2l2 bi-allelic mutation is associated with mice that are phenotypically normal except for male sterility (Print et al. 1998). This results from abnormal supporting Sertoli cell architecture, and is rescued by loss of Bax but not Bak (Print et al. 1998; Ross et al. 2001).

1.4.1.2.6 BCL-B

Cloning of BCL-2-like protein 10 (BCL-B, Boo or Diva) revealed that it contains the BH1 and BH2 domains and a comparable BH4 domain conferring similarity to other BCL-2 family members, though unlike the others BCL-B lacks the BH3 domain (Song et al. 1999). Extension of these studies revealed further similarity to BCL-W in its restricted tissue expression, being expressed mostly in ovarian tissue and required for spermatogenesis.

1.4.1.3 BH3-only proteins

Unlike the effector proteins or anti-apoptotic family members, the BH3-only proteins only express the BH3 and transmembrane domains (Figure 1.7). In many different cellular contexts, the transcriptional or translational upregulation of these proteins following exposure to stressors is the initiating event in altering the equilibrium between pro-death and pro-survival components leading to apoptosis (Adams and Cory 2007). The BH3-only proteins then use the BH3 domain to bind the hydrophobic groove within the anti-apoptotic proteins, and in doing so release

51 Chapter 1 the effector proteins from this site (Adams and Cory 2007; Czabotar et al. 2013; 2014; Dai et al. 2011; Westphal et al. 2013). Similarly, the BH3-only proteins engage the effector proteins directly to induce activating conformational changes leading to their homo-oligomerization (Czabotar et al. 2013).

1.4.1.3.1 BIM

Protein binding studies have shown BCL-2-like protein 11 (BIM) to interact with BCL2L11 all of the anti-apoptotic proteins with high affinity (Chen et al. 2005a). elevation in blood lymphocyte and granulocyte numbers, leading to autoimmunity genetic deletion studies in mice have shown significant haematological effects with and renal failure (Bouillet et al. 1999; Herold et al. 2014). Mutation of BCL2L11 is not frequently described in cancer, though genetic studies using the Eµ-Myc

2004) and a common deletional genetic polymorphism of BIM has been associated lymphoma model have confirmed BIM to be a tumour suppressor (Egle et al. with refractoriness to tyrosine kinase inhibitor treatment of chronic myelogenous leukaemia (Ng et al. 2012).

1.4.1.3.2 BID

BH3 interacting-domain death agonist (BID) is distinct from other BH3-only proteins in that it is located in the cytosol and cleaved by caspase 8 to a truncated function (McDonnell et al. 1999). This unique interaction between caspase 8 and form (tBID) that then localises to the mitochondria to fulfil its BH3-only protein BID allows for interplay between the intrinsic and extrinsic pathways, providing

Similar to BIM, tBID is also highly promiscuous in that it binds all anti-apoptotic significant amplification of apoptosis upon activation of the extrinsic pathway. proteins (Chen et al. 2005a). Bid knockdown can rescue many of the phenotypic Bid has been shown to abrogate development of FAS ligand-induced hepatocellular carcinoma aberrations of Bcl-2 deficiency in mice, and tissue-specific deletion of (Ni et al. 2008; Bai et al. 2010).

1.4.1.3.3 PUMA

The p53 upregulated modulator of apoptosis (PUMA, also known as BCL2-binding component 3) is encoded by the BBC3 gene on chromosome 19, the deletion of which has been described as a recurrent event in cancer (Beroukhim et al. 2010). PUMA readily binds all BCL-2 family members and its overexpression confers the greatest degree of apoptosis to murine embryonic fibroblasts (MEFs) 52 Chapter 1 when compared to overexpression of the other BH3-only proteins (Chen et al. 2005a). PUMA and NOXA are distinct from other BH3-only proteins in that they are transcriptional targets of p53 (Nakano and Vousden 2001). Critically, upon deletion of Puma and Noxa, cells become resistant to apoptosis induction following suppressor functions (Michalak et al. 2008). Of relevance to current lymphoma DNA damaging stimuli including cytotoxic therapies, confirming their tumour treatment with prednisolone, PUMA is a mediator of glucocorticoid activity via a p53-independent mechanism (Villunger et al. 2003).

1.4.1.3.4 NOXA

Phorbol-12-myristate-13-acetate-induced protein 1 is better known as NOXA and is encoded by the PMAIP1 gene on chromosome 18. NOXA selectively binds MCL-1 and A1, is an effector of p53-induced apoptosis, and its knockdown is protective against apoptosis induction of MEFs exposed to cytotoxic insults (Chen et al. 2005a; Nakano and Vousden 2001) suppressor protein. . These findings confirmed its role as a tumour

1.4.1.3.5 BAD

BCL-2-associated death promoter (BAD) selectively binds BCL-2, BCL-XL and BCL-W, but not MCL-1 or A1 (Chen et al. 2005a). Bad-/- mice are phenotypically normal with the exception of subtle lymphocyte abnormalities, however, upon aging they have been shown to develop lymphoma phenotypically similar to DLBCL, and upon exposure to irradiation they acquire precursor B- and T-cell neoplasms (Ranger et al. 2003).

1.4.1.3.6 BMF

BCL-2-modifying factor (BMF) binds all BCL-2 family members, though has a higher

L and BCL-W than with MCL-1 or A1 (Chen et al. 2005a). Bmf-/- mice are phenotypically normal with the exception of expansion affinity for association with BCL-2, BCL-X of the B-lymphocyte population (Labi et al. 2008). Interestingly, lymphocytes from these mice are protected from glucocorticoid-mediated apoptosis, but not from other cytotoxic stimuli. Finally, when these mice were exposed to irradiation, they also showed a propensity for development of T-cell lymphoma.

53 Chapter 1

1.4.1.3.7 BIK

BCL-2-interacting killer (BIK) binds all of the BCL-2 family members, though interestingly its overexpression in MEFs is not associated with potent apoptosis induction (Chen et al. 2005a). Despite broad tissue expression of Bik, Bik-/- mice are phenotypically normal and knockout of Bik effects of Bcl2 deletion (Coultas et al. 2004). Of interest, however, BIK deletion is insufficient to rescue the phenotypic suppressor function (Sturm et al. 2005). is a frequent and specific event to renal cell carcinoma, confirming a tumour

1.4.1.3.8 HRK

Despite interacting with all of the BCL-2 family members, forced overexpression of activator of apoptosis, harakiri (HRK) does not induce apoptosis of MEFs (Chen et al. 2005a). HRK is restricted in its expression to neuronal tissue and its deletion confers minimal change to apoptosis (Coultas et al. 2007). Hence, despite its BH3- only structure, no definitive oncogenic role has been described for HRK. 1.4.1.4 Downstream effectors of apoptosis induction

The downstream cascade of apoptosis is initiated following release and activation of BAX / BAK, homo-oligomerisation and perforation of the MOM. The damaged mitochondrion then releases cytochrome c into the cytoplasm where it binds APAF-1, which itself binds caspase-9 through NH2-terminal interactions (Figure 1.6) (Zou et al. 1997; Cecconi et al. 1998). The presence of cytochrome c and dATP at this interaction results in full activation of caspase-9, which in turn cleaves and activates caspase-3 (Li et al. 1997). Notably, deletion of Apaf-1 in mice is embryonic lethal with significant malformation of the face, brain, eyes and digits plays in apoptosis (Cecconi et al. 1998). due to a global impairment in apoptosis, confirming the critical role this process

Following its activation, caspase-3 cleaves and activates caspases-6 and -7, then cleave a large number of proteins at the C-terminus of aspartic acids, causing allowing for further amplification of the downstream cascade. Activated caspases destruction of the target proteins and in turn ceasing a variety of critical cellular processes with resultant cell death (McIlwain et al. 2013).

54 Chapter 1

Regulation of the downstream cascade must occur to ensure that caspase activation release. In the steady state this repression is mediated by XIAP (Du et al. 2000). is sufficiently repressed prior to the activating signal through cytochrome c Following release of cytochrome c and concomitant with activation of caspases, the mitochondrion also releases second mitochondria-derived activator of caspases (SMAC). SMAC represses the activity of XIAP, removing its repression of caspase activation and in turn further enhancing the downstream signalling cascade leading to cell death (Du et al. 2000). While it may therefore appear that SMAC-mimetics could be useful tools in cancer therapy, our group has recently shown that use of SMAC-mimetics in Eµ-Myc lymphoma leads to disease acceleration concomitant with cytokine release and susceptibility to endotoxic shock (West et al. 2016).

1.4.2 Extrinsic apoptosis

the hypotheses and experiments described in this thesis. This pathway mediates The extrinsic apoptosis pathway will only be discussed briefly as it is not central to extracellular signals by ligand engagement of death receptors, which are members of the tumour necrosis factor (TNF)-receptor family. Upon activation of these receptors the intracellular components of the receptor mediate signalling through the FAS-associated protein with (FADD), leading to cleavage and release of caspase-8 (Strasser et al. 2009). Caspase-8 then cleaves and activates caspases-3 and -7, and may itself also perform catalytic cleavage of target proteins critical for cell survival. Furthermore, capsase-8 cleaves BID to produce its active form tBID, providing a critical interplay with the intrinsic apoptotic pathway and further amplifying cell death signalling (McDonnell et al. 1999).

1.4.3 Small molecule inhibition of BCL-2 family members

Given the equilibrium maintained between pro-apoptotic and anti-apoptotic proteins, it is logical that targeting the interaction of these proteins may provide a useful approach to restoring death to protected cancer cells. However, as opposed to the relative ease of targeting a kinase domain to switch off oncogenic processes, required exhaustive efforts of medicinal chemistry to slowly advance toward the the affinity of protein-protein interactions is markedly increased and therefore has compounds emerging in the clinic today (D. Huang 2016, personal communication, 13 April).

55 Chapter 1

As the structures of BH3-only proteins have been described, the logical approach to restoring apoptosis has been the development of ‘BH3-mimetics’. These compounds mimic BH3-only proteins, leading to displacement of BAX/BAK and their subsequent activation leading to perforation on the MOM. While a number of preclinical compounds have provided useful tools for interrogating the differing interactions of apoptosis family members and the critical dependencies of many cancer types, only recently have these compounds become established in the clinic as evidenced by FDA approval of the BCL-2-antagonist, venetoclax (ABT-199), in April 2016 for the treatment of CLL (Souers et al. 2013). Precursor compounds that targeted BCL-2, BCL-XL and BCL-W, such as ABT-263 (navitoclax) and ABT- 737, showed clinical activity against select neoplasms but at the expense of dose- limiting thrombocytopenia mediated by BCL-XL inhibition (Mason et al. 2008; 2007). Despite the ability to produce compounds targeting these select BCL-2 family members, direct targeting of MCL-1 has until recently proven elusive (Souers et al. 2013; Leverson et al. 2014; Xiao et al. 2015). At the time of preparation of this thesis, there are no studies describing in vivo activity of any putative small molecule BH3-mimetic targeting MCL-1.

1.5 Conclusions and hypothesis

There is a clear need for novel therapeutic approaches to overcome the resistance of aggressive B-cell lymphoma, particularly in the relapsed and refractory setting. MYC is frequently dysregulated in this disease group, and appears to associate with aggressive disease phenotypes characterised by rapid cellular proliferation and instability as demonstrated by the pathological characteristics of Burkitt lymphoma.

It is hypothesised that MYC dysregulation confers a state of ‘transcriptional addiction’ to aggressive malignancies, and as MYC itself has proven an elusive therapeutic target, perhaps short-lived proteins that are transcriptional targets of MYC would provide indirect means through which to counter its activity. Anti- apoptotic MCL-1 presents an attractive therapeutic target due to its extremely short half-life and the historical associations of its transcriptional repression MCL1 deletion and the phenotype of Mcl1-/- mice have suggested a critical dependency of through use of first generation pan-CDK inhibitors. Furthermore, studies of haematopoietic cells upon Mcl-1 expression.

The key aims of this thesis were to use genetic and pharmacologic tools to interrogate whether CDK9 activation of Pol II transcription is an oncogenic

56 Chapter 1 dependency of MYC-dysregulated B-cell lymphoma, and the role that MCL-1 plays as a downstream effector of this process (Figure 1.8). It is hypothesised that MYC, CDK9 and MCL-1 represent a druggable linear pathway and that pharmacological inhibition of CDK9 or MCL-1 would provide an effective novel therapeutic strategy to overcome the resistance of this disease group.

P-TEFb Cyclin T1 CDK9 MYC YSPTSPS/YSPTSPSCTD RNA polymerase II

Transcription

MCL-1

Figure 1.8: Proposed ‘linear pathway’ involving CDK9 regulation of Pol II- mediated transcription of critical MYC targets including MCL-1

Schematic representation of the proposed oncogenic pathway demonstrating MYC recruitment of cyclin T1 to recruit P-TEFb to MYC transcriptional target sites. CDK9 phosphorylates RNA Polymerase II, subunit B1 at serine residues at position two within heptapeptide repeats in the carboxy-terminal domain (CTD), leading to transcription of critical effector proteins such as MCL-1. P denotes phosphorylation.

57 58 Chapter 2: Materials & Methods

59 Chapter 2

2.1 Tissue culture

2.1.1 Eµ-Myc lymphoma cell culture

Spontaneously generated and derived Eµ-Myc lymphoma cell lines (Table 2.1) were sourced from the Johnstone cell bank, Peter MacCallum Cancer Centre (PMCC, Victoria Australia). Lymphomas were cultured in treated six-well tissue culture plates (CELLSTAR®, Greiner Bio-One, Frickenhausen, Germany) with Anne referred to as Eµ-Myc Media (EMM). Tissue culture was performed at 37°C and Kelso modification of Dulbecco’s modified Eagle’s medium (DMEM), commonly 10% CO2 atmospheric condition with maintenance of exponential growth through passaging three times per week.

2.1.1.1 Anne Kelso Modified DMEM

with glucose 4g/L, folic Acid 6mg/L, L-Asparagine 36mg/L (Sigma-Aldrich®, St DMEM, low glucose (Thermo Fisher Scientific™, Waltham, MA, USA) supplemented Louis, MO, USA), L-Arginine HCl 116mg/L (Sigma-Aldrich®, St Louis, MO, USA), ® L-Glutamine 216mg/L (Sigma-Aldrich , St Louis, MO, USA), NaHCO3 2g/L and HEPES 10mM (Merck Millipore®, Billerica, MA, USA).

2.1.1.2 Eµ-Myc Media (EMM)

serum (FBS) (Sigma-Aldrich®, St Louis, MO, USA), penicillin / streptomycin 2mM Anne Kelso Modified DMEM supplemented with 10% heat inactivated foetal bovine

Aldrich®, St Louis, MO, USA) and 2-Mercaptoethanol 50µM (Sigma-Aldrich®, St (Thermo Fisher Scientific™, Waltham, MA, USA), L-Asparagine 100µM (Sigma- Louis, MO, USA).

2.1.2 Human IG-MYC-translocated cell culture

BL-41, Ramos, Raji, Namalwa, OPM2 and H929 cell lines were sourced from the Cancer Immunology cell bank, PMCC. Lymphomas were cultured in six-well tissue culture plates (CELLSTAR®, Greiner Bio-One, Frickenhausen, Germany) or 25cm2 ®, Greiner Bio-One, Frickenhausen, Germany) in RPMI-

flasks (CELLSTAR heat inactivated FBS (Sigma-Aldrich®, St Louis, MO, USA) and 2mM penicillin / 1640 (Thermo Fisher Scientific™, Waltham, MA, USA) supplemented with 10% °C and 5% CO atmospheric condition. streptomycin2 (Thermo Fisher Scientific™, Waltham, MA, USA) at 37

60 Chapter 2

Name Background Alteration Source Eµ-Myc #20 C57BL/6 x Bmf-/- Johnstone bank #30 C57BL/6 x Bim-/- Johnstone bank #106 C57BL/6 p53 mutant Johnstone bank #107 C57BL/6 Johnstone bank * #3391 C57BL/6 x p53-/- Johnstone bank #4242 C57BL/6 Johnstone bank MSCV Johnstone bank MSCVtmBcl-2 Johnstone bank MSCVtmMcl-1 Johnstone bank

MSCVtmBcl-XL Johnstone bank MSCVthBCL-2 G. Gregory MSCVthMCL-1 G. Gregory

MSCVthBCL-XL G. Gregory MSCVthA1 G. Gregory MSCVthBCL-W G. Gregory REBIR.shSrambled G. Gregory REBIR.shCdk9.421 G. Gregory REBIR.shCdk9.2869 G. Gregory REBIR.shMcl-1.1792 G. Gregory #6066 C57BL/6 NrasQ61K mutation Johnstone bank TRMPVIR.p53 Johnstone bank *

Vκ*Myc #4929 C57BL/6 MSCV Johnstone bank

Table 2.1: Characteristics of Eµ-Myc lymphomas and Vκ*Myc multiple myeloma used in experimental work

mus musculus; h, homo sapiens. *Denotes spontaneously arising lesion; MSCV, murine stem cell virus; m,

61 Chapter 2

2.1.3 Cell lines for retroviral and lentiviral use

NIH3T3 target cells and HEK293T packaging cells were sourced from the Cancer Immunology cell bank, PMCC. Cells were cultured in 175cm2 ®, Greiner Bio-One, Frickenhausen, Germany) in SAFC DMEM supplemented with flasks (CELLSTAR 10% heat inactivated FBS (Sigma-Aldrich®, St Louis, MO, USA) and 2mM penicillin °C and 10% CO atmospheric condition. / streptomycin2 (Thermo Fisher Scientific™, Waltham, MA, USA) at 37

2.1.4 Compounds for in vitro use

Cytotoxic and targeted chemical compounds used for in vitro studies are listed in Table 2.2. Dinaciclib, A1592668.1, palbociclib, JQ1 and ABT-199 were reconstituted in DMSO (Merck Millipore® stored at -20°C and thawed a maximum of three times per aliquot. AZ-CDK9, , Billerica, MA, USA) at a final concentration of 10mM, AZ-MCL1 and AZD4320 were reconstituted in DMSO (Merck Millipore®, Billerica, a dessicator. Aliquots removed from the dessicator were used for only a period MA, USA) for a final concentration of 10mM and stored at room temperature in of one week then discarded. Flavopiridol was reconstituted in sterile water for °C and thawed a maximum of three times per aliquot. Etoposide (sourced from clinical pharmacy stocks, PMCC) was a final concentration of 5mM, stored at -20 reconstituted directly into media prior to use.

2.2 Flow cytometry apoptosis and cell cycle assays

2.2.1 Cell Death assays

Cells in exponential growth phase (2 x 105) were cultured in 24-well tissue culture plates (CELLSTAR®, Greiner Bio-One, Frickenhausen, Germany) in the presence of various concentrations of chemical compound in 400µL EMM or RPMI with additives, according to cell type. Untreated and DMSO-treated wells (equal to maximum concentration of compound) were included as controls for the diluent of compound. Following incubation for the desired time period, cells were gently spun to create cell pellets, the supernatant was discarded and the cells were resuspended in the appropriate buffer and fluorochrome-labelled antibody for (Franklin Lakes, NJ, USA) unless otherwise stated. assessment. Flow cytometric analysis was performed on the BD FACSCanto™ II

62 Chapter 2

Chemical compound Source Vehicle (in vitro) A1592668.1 AbbVie Inc., DMSO Boston, MA, USA ABT-199 AbbVie Inc., DMSO Boston, MA, USA AZ-CDK9 AstraZeneca, DMSO Waltham, MA, USA AZ-MCL1 AstraZeneca, DMSO Waltham, MA, USA AZD4320 AstraZeneca, DMSO Waltham, MA, USA Dinaciclib Merck, DMSO Boston, MA, USA Etoposide PMCC cytotoxic suite, DMSO Victoria, Australia

Flavopiridol Selleck Chemicals, H2O Houston, TX, USA JQ1 J. Bradner, DMSO Boston, MA, USA Palbociclib Shatha Abuhammad, DMSO PMCC, Victoria, Australia

Table 2.2: Chemical compounds described in in vitro studies

DMSO, dimethylsulfoxide

63 Chapter 2

2.2.1.1 Annexin-V and propidium iodide apoptosis assays

Plasma membrane disruption was assessed by phosphatidyl-serine externalisation permeabilisation using propidium iodide (PI) 69mM (Sigma-Aldrich®, St Louis, using annexin-V-APC (BD Pharmingen™, San Jose, CA, USA) binding and cell MO, USA) following preparation at 1:100 volume/volume (v/v) dilution in annexin binding buffer comprising HEPES 10mM (pH 7.4), NaCl 140mM and CaCl2 5mM. Following 10 minutes of incubation, cells positively staining for annexin-V and PI were defined as apoptotic. 2.2.1.2 Mitochondrial outer membrane potential assessment (TMRE)

Mitochrondrial outer membrane permeabilisation was assessed following 10 minutes incubation with tetramethylrhodamine ethyl ester perchlorate (TMRE, Molecular Probes, Eugene OR, USA) at a dilution of 1:10,000 in PBS. Cells lacking apoptotic. TMRE staining (compared to unstained and untreated controls) were defined as

2.2.1.3 Cell cycle analysis according to nuclear DNA content

For cell cycle analysis, cells were resuspended in 100µL Nicoletti buffer comprising

PI 50mg/ml, sodium citrate 0.1% (pH 7.4) and Triton X-100 0.1%. Following flow cytometric analysis, gating was performed manually using Flowlogic™ software as those in the sub-G1 gate. (Inivai technologies, Mentone, VIC, Australia) and the apoptotic cells were defined

2.3 Protein techniques

2.3.1 Preparation of protein

For in vitro specimens, cells in media were harvested into 10mL falcon tubes (Sarstedt AG & Co., Nümbrecht, Germany) and centrifuged 1,400 rpm at 4°C for four minutes prior to discarding supernatant. Cell pellets were resuspended in 1mL ice cold PBS and transferred to microcentrifuge tubes, prior to microcentifugation at 5,000rpm for two minutes to pellet cells and discard supernatant. Cells were then resuspended and lysed in RIPA lysis buffer comprising 50mM Tris (pH 8), 150mM NaCl, 1% NP-40, 0.5% Na-deoxycholate and 0.1% SDS, supplemented with cOmplete™, Mini, EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics GmbH, Mannheim, Germany) and phosSTOP™ phosphatase inhibitor 64 Chapter 2 cocktail tablets (Roche Diagnostics GmbH, Mannheim, Germany). Following 30 minute incubation on ice, lysates were microcentrifuged at 13,000 rpm, 4°C for 15 minutes to clear insoluble cellular debris and the supernatant transferred to another set of microcentrifuge tubes stored on ice.

19µL PBS, from which 5µL was placed into a 96-well round bottom plate (CELLSTAR®, Protein quantification was next performed following dilution of 1µL of lysate with Greiner Bio-One, Frickenhausen, Germany) and 195µL Coomassie protein assay performed using the Bradford method and standard controls of bovine serum reagent (Thermo Scientific, Rockford, IL, USA) was added. Quantification was

Versamax Microplate reader and analysis with SoftMax Pro software version 5.4 albumin (Thermo Scientific, Rockford, IL, USA) via absorbance at 595 nm using a (Molecular Devices, Sunnyvale, CA, USA). Lysates were then normalised to 1µg/µL with the addition of UltraPure™ DNase/RNase-free distilled water (Thermo Fisher (250mM Tris-HCl [pH 6.8], glycerol 50% v/v, 10% SDS weight/volume [w/v], 2.5% Scientific™, Waltham, MA, USA) and diluted in 5x storage buffer with loading dye 2-mercaptoethanol v/v and 0.001% bromophenol blue w/v) prior to storage at -80°C.

2.3.2 Protein separation, transfer and immunoblotting

Cell lysates were heated at 95° polyacrylamide gel electrophoresis using 4-20% gradient polyacrylamide precast C for five minutes prior to separation by gels (Mini-PROTEAN® (25mM Tris-HCl, 192mM glycine, 0.1% SDS w/v in milli-Q H 0) with 5µL molecular TGX™ gel, Bio-Rad, Hercules, CA, USA)2 in SDS running buffer

Rockford, IL, USA). Protein was transferred from gels to Immobilon-P PVDF weight standard (PageRuler™ Prestained Protein Ladder, Thermo Scientific, membrane (Millipore, Bedford, MA, USA), through electroblotting with transfer apparatus (Bio-Rad, Hercules, CA, USA) using wet transfer buffer (25mM Tris-HCl [pH 8.3], 192mM glycine, 15% methanol v/v) at 100V for 90 minutes at 4°C. For high molecular weight transfers (>150 kDa), the protocol was adapted to use 5% methanol v/v and transfer for 120 minutes.

Membranes were then blocked through one-hour incubation in 5% skim milk membranes were incubated for 18-24 hours at 4°C with primary antibody (Table powder w/v in PBS. Following three wash steps with PBS for five minutes each, 2.3 were incubated with the cognate secondary antibody (HRP-conjugated) (Table ). Following three further wash steps with PBS for five minutes each, membranes 2.3

) prior to three further five-minute wash steps in PBS. Membranes were then 65 Chapter 2 ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ) ! ! ! 9$4;#$&" AB5CCC!<4D*('8@ AB5CCC!<4D*('8@ AB5CCC!<4D*('8@ AB5CCC!<4D*('8@ AB4CCC!<4D!3E;@ AB4CCC!<4D!3E;@ ABACCC!<4D!3E;@ ABACCC!<4D!3E;@ ABACCC!<4D!3E;@ ABACCC!<4D!3E;@ ABACCC!<4D!3E;@ ABACCC!<4D!3E;@ ABACCC!<4D!3E;@ ABACCC!<4D!3E;@ ABACCC!<4D!3E;@ AB4CCC!<4D!3E;@ ABACCC!<4D!3E;@ ABACCC!<4D!3E;@ ABACCC!<4D!3E;@ ABACCC!<4D!3E;@ ABACCC!<4D!3E;@ ABACCC!<4D!3E;@ ! 4CS ! RZGE SRA ! # ! ! ! ! ! ! ! ! ! ! ! ! ! RRC ! SCG # GCA # EU; ) .4&"- # # # NG ! 4CZ ! SRZ5 ! 5C5N ! # # # # ! =*A; ;M QRSAG 44G5AS ! E7 E7 ;;U ;JY QZGC5 Q5RAT Q445G ;=[ QARGZZ QGNR4 QAR45R QZ4R5 TCC # QG4N5 E7 ! 44GCA5 ! UCATA UCG4C UC5AN ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ) *K1!LE;@ ! ! ! 51";<12#;/-/ '()H!<=0)P-6,1!*;1!LE;@ '()H!<=0)P-6,1!*;1!LE;@ '()H!<=0)P-6,1!*;1!LE;@ '()H!<=0)P-6,1!*;1!LE;@ '()H!<=0)P-6,1!*;1!LE;@ '()H!<=0)P-6,1!*;1!LE;@ '()H!<=0)P-6,1!*;1!LE;@ '()H! <=0)P-6,1!*;1!LE;@ '()H!<=0)P-6,1!*;1!LE;@ )0 )0 )0 )0 )0 )0 )0 )0 )0 ;'>6(7I!$1!:-6/0)?@ ! E(H/0 M-''!E(H 3=!UI06/()H-)!0'-1!_`1!LE;@ M-''!E(H M-''!E(H M-''!E(H ])W+!JE!<^06/()H>0'-1!_`1!LE;@ M-''!E(H M-''!E(H M-''!E(H M-''!E(H O+78'0)>!<:('&-6$,P(''-1!U;1!LE;@ M-''!E(H E0)$0!M6%W!<=0''0,1!XY1!LE;@ ! 3=!UI06/()H-)!?@ A))B/$C1/()1"')+-2&"'1/()1"#$%&'$-+ *21!/+'-7%'06!c-(HI$d!8=01!8('+>0'$+)d!231!2-,$-6)!&'+$d!3E;1!& ! !

66 Chapter 2 visualised for antibody expression using HRP-substrate chemiluminescent reagent

Little Chalfont, Buckinghamshire, UK) and developed on RX-N Fuji Medical X-Ray (Amersham™ ECL™ Western Blotting Detection Reagents, GE Healthcare UK Ltd.,

Belgium). The above process was repeated with loading control antibodies Film (Fujifilm, Tokyo, Japan) using the Agfa CP1000 developer (Agfa, Mortsel, (β-actin, α-tubulin or HSP90) for all membranes.

2.4 Ribonucleic acid techniques

2.4.1 Preparation of RNA

Following in vitro culture, cells in media were harvested into 10mL falcon tubes (Sarstedt AG & Co., Nümbrecht, Germany) and centrifuged 1,400rpm at 4°C for four minutes prior to discarding of supernatant. The cell pellets were resuspended in 1mL ice cold PBS and transferred to microcentrifuge tubes, prior to microcentifugation at 5,000rpm for two minutes to pellet cells and discard supernatant. RNA was extracted using the Nucleospin® RNA isolation

(Macherey-Nagel, Bethlehem, PA, USA) and eluted in 40µL UltraPure™ DNase/ RNase-free distilled water (Thermo Fisher Scientific™, Waltham, MA, USA). 2.4.2 Generation of complementary DNA

RNA quantification was performed on the Nanodrop 2000 (Thermo Fisher 1µg RNA in 11.5µL DNase/RNase-free distilled water, then the addition of 0.5µL Scientific™, Waltham, MA, USA). Complementary DNA (cDNA) was produced using random primers (Promega, Sydney, Australia) and heated at 70° then cooled to 5°C. Next, a mastermix comprising MMLV (5x) 5µL, 0.1mM DTT C for five minutes 2.5µL, 10mM dNTPs 1.25µL, RNase inhibitors 0.25µL, reverse transcriptase 0.25µL (Promega, Sydney, Australia) and DNase/RNase-free distilled water 11.25µL was added to each sample prior to incubation for 50 minutes at 42° at 95°C to terminate the reaction. C then five minutes

2.4.3 Quantitative reverse-transcription PCR

A master mix was prepared for each target gene set comprising cDNA 1.5µL, DNase/RNase-free distilled water (10.5µL), and SYBR green 14µL (Invitrogen, Life Technologies, Victoria, Australia). Into a 384 well plate (Applied Biosystems, Melbourne, Australia) were applied 8µL of mastermix mix in triplicate to which 2µL of forward / reverse primers targeting the exon-exon boundaries of target

67 Chapter 2 genes were added (Table 2.4

) and amplification performed on an ABI4900 light 95°C for 30 seconds alternating with 60°C for 30 seconds for 40 cycles and a cycler (Thermo Fisher Scientific™, Waltham, MA, USA) with PCR conditions of GAPDH for murine specimens and L32 for human specimens. final dissociation cycle. Housekeeping genes were included as controls;

2.4.3.1 qRT-PCR statistical analysis

enabled expression of the gene as a fold change related to a separate housekeeping Each sample amplification was performed in triplicate and the final analysis control gene and background. This calculation was performed in four parts. Firstly, the delta CT (ΔCT) value (control gene) was calculated by subtracting the CT (background) from the CT (control gene). Next, the delta CT value (target gene) was calculated by subtracting the CT (background) from the CT (target gene). Next, the delta delta CT (ΔΔCT, target to background) was performed by calculation. Finally, the fold change was calculated as 2ΔΔCT. subtracting the figure from the first calculation from the figure from the second

2.5 Chromatin immunoprecipitation

2.5.1 Preparation of immunoprecipitated samples

Formaldehyde 1% v/v was added to 1 x 107 cells in media per sample and placed on an orbital shaker at 40rpm at room temperature for 15 minutes. Glycine 0.125M washed twice with cold PBS and transferred to microcentrifuge tubes. Cells were v/v was added for five further minutes. Cells were harvested, centrifuged and resuspended in 300µL ChIP lysis buffer (1% SDS v/v, 10mM EDTA, 50mM TRIS-

HCl [pH 8], supplemented with cOmplete™, Mini, EDTA-free protease inhibitor phosphatase inhibitor cocktail tablets [Roche Diagnostics GmbH, Mannheim, cocktail tablets [Roche Diagnostics GmbH, Mannheim, Germany] and phosSTOP™ Germany]) prior to sonication with the Covaris S2 (Covaris Inc., Woburn, MA, USA) set to 30 seconds on and 30 seconds off for a total of 35 minutes. Lysates were spun

(1% Triton X-100 v/v, 0.1% sodium deoxycholate w/v, 0.1% SDS w/v, 90mM NaCl, at 13,000rpm then immunoprecipitation was performed in modified RIPA buffer inhibitor cocktail tablets [Roche Diagnostics GmbH, Mannheim, Germany]). Input 10mM TRIS-HCl [pH 8], supplemented with cOmplete™, Mini, EDTA-free protease sample (4%) was removed and antibody (1:100 v/v) was added prior to overnight incubation at 4°C. Protein A beads (50µL, nProtein A Sepharose 4 Fast Flow, GE Healthcare, Feiburg, Germany) were added the next day and samples were

68 Chapter 2

Primer Sequence (5’ à 3’) Mus musculus Bcl-2 F: ATGACTGAGTACCTGAACCGGCAT R: GGGCCATATAGTTCCACAAAGGCA

Mcl-1 F: GGTGCCTTTGTGGCCAAACACTTA R: ACCCATCCCAGCCTCTTTGTTTGA

GAPDH F: CCTTCATTGACCTCAACTAC R: GGAAGGCCATGCCAGTGAGC

Homo sapiens cMYC F: GGACGACGAGACCTTCATCAA R: CCAGCTTCTCTGAGACGAGCTT

MCL-1 F: AACAAAGAGGCTGGGATGGGTTTG R: AAACCAGCTCCTACTCCAGCAACA

L32 F: TTCCTGGTCCACAATGTCAAG R: TTGTGAGCGATCTCGGCAC

ChIP Mcl-1 Set 1 F: TTCCTCACTCCTGACTTCCG R: CCAAACATGGTCGGACGC

Mcl-1 Set 2 F: TGTAAGGACGAAACGGGACT R: CACCCCATTTCCACTCCACG

Mcl-1 Set 3 F: TAGAGATGGAAGAGGGGCCAG R: TAGGGCTTCTCTCTCAACACTC

Table 2.4: Primer sequences used for qRT-PCR and ChIP experimentation

F, Forward; R, Reverse; ChIP, chromatin immunoprecipitation

69 Chapter 2 incubated at 4°C for two hours. Beads were washed twice with 1mL ChIP wash buffer (0.1% SDS v/v, 1% Triton X-100 v/v, 2mM EDTA, 150mM NaCl and 20mM

Triton X-100 v/v, 2mM EDTA, 500mM NaCl and 20mM TRIS-HCl [pH 8]), then TRIS-HCl [pH 8]) prior to a wash with ChIP final wash buffer (0.1% SDS v/v, 1% elution for 15 minutes in 240µL elution buffer (1% SDS, 0.1M NaHCO3). RNase 2µL per sample was added prior to overnight de-crosslinking at 65°C.

2.5.2 Separation, extraction and quantitation of DNA

® PCR Clean-up kit (Macherey-

De-crosslinked DNA was purified using the Nucleospin Nagel, Bethlehem, PA, USA) and eluted into 20µL UltraPure™ DNase/RNase-free performed using qRT-PCR as described above. distilled water (Thermo Fisher Scientific™, Waltham, MA, USA). Quantitation was

2.6 Retroviral transduction

2.6.1 Retroviral supernatant production

Retroviral transduction was performed using standard calcium phosphate 6 HEK293T cells were plated in 10cm round tissue culture plates (CELLSTAR®, Greiner Bio-One, Frickenhausen, Germany) 24 transfection techniques. Briefly, 3 x 10 hours prior to transfection and a media change performed three hours prior to transfection. Plasmid DNA (10µg) and amphotrophic virus (10µg) were prepared in a solution containing 2.5M CaCl2 / HEPES 1.25mM and added to equal volume of 2x HEPES buffered saline (HEPES 0.05M, NaCl 0.28M, Na2HPO4 1.5mM [pH 7]) concomitant to air bubbles to assist calcium precipitate formation. Following 20-minute incubation at room temperature, the solution was added drop-wise to the HEK293T cells and mixed with gentle agitation. A media change followed 16 hours later (to target cell media) and cells continued to culture for another 24

(Acrodisc®, PALL Life Sciences, Port Washington, NY, USA) to prevent transfer of hours prior to harvesting of virus-containing supernatant through 0.45µm filters cellular debris. Fresh media was reapplied and the process repeated 24 hours later to harvest further viral supernatant.

2.6.1.1 Retroviral transduction of Eµ-Myc lymphoma cells

Six-well non-treated tissue culture plates (Costar®, Kennebunk, ME, USA) were coated with RetroNectin® 3µg/mL (Takara Bio Inc., Madison, WI, USA) in PBS for two °C. hours prior to replacement with 1mL 5% filter-sterilised BSA for storage at 4 70 Chapter 2

BSA was removed prior to addition of the viral supernatant and centrifugation of plates at 2000x G for 60 minutes at room temperature. Viral supernatant was removed and 1 x 106 target cells in 4mL media were added then incubated for 24 ° hours at 37 C at 10% CO2 atmospheric condition. The following day, a second set of plates were prepared, to which viral supernatant was added as per previous. in 4mL media and added to the second set of plates for a second transduction The same target cells were then harvested from the first set of plates, resuspended protein positive expression and returned to exponential growth culture for in vitro exposure. Following 72 hours incubation, cells were FACS-sorted for fluorescent- interrogation.

2.6.2 Lentiviral transduction

2.6.2.1 Lentiviral supernatant production

Lentiviral constructs were sourced from the Victorian Centre for Functional Genomics (PMCC, Victoria, Australia) and the sequences are listed in Table 2.5. HEK293T cells were transfected using standard polyethylenimine (PEI) 6 HEK293T cells were plated into a single well of a six-well tissue culture plate (CELLSTAR®, Greiner Bio-One, Frickenhausen, transfection techniques. Briefly, 1.5 x 10 Germany) and cultured in DMEM supplemented with 10% tetracycline-free FBS at ° 37 C at 10% CO2 atmospheric condition. The following day cells were inspected mix 5µL (Victorian Centre for Functional Genomics, Victoria, Australia), pGIPZ and virtually 100% confluent. A mastermix comprising Lenti-X HTX packaging construct 2µg and tetracycline-free FBS 170µL was vortexed, prior to addition of 20.3µL of PEI (1mg/mL stock) and repeat vortex prior to 10 minute incubation at room temperature. The mix was then re-vortexed prior to dropwise addition to cells in culture and gentle agitation of media to disperse the lentivirus. Cells ° were incubated at 37 C and 10% CO2 atmospheric condition for 24 hours prior to media change to RPMI supplemented with 10% tetracycline-free FBS. The following day, viral supernatant was harvested, spun at 1,400rpm for four minutes to pellet cellular debris, then the supernatant was aliquoted into microcentrifuge tubes and stored at -80°C.

2.6.2.2 Lentiviral transduction of OPM2 cells

OPM2 cells were cultured in exponential growth phase and seeded at 20,000 cells per well of a 96-well round bottom tissue culture plate (CELLSTAR®, Greiner Bio-One, Frickenhausen, Germany) in 100µL RPMI supplemented with 10%

71 Chapter 2

! "#$#! %#&'#$(#! "#$#! %#&'#$(#!

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

Table 2.5: Lentiviral construct sequences

72 Chapter 2

Target Sequence Cdk9.421 TCAAACACCAGATAGATGCTGC Cdk9.2869 TAGTTTGTCTTCATGCCACTGA Mcl1.1334 AAAGAGTCACTGTCTGAATG Mcl1.1792 AAACAGCCTCGATTTTTAAG Mcl1.2018 CGGACTGGTTATAGATTTAT

Table 2.6: Short-hairpin RNA sequences targeting Cdk9 and Mcl1

73 Chapter 2 tetracycline-free FBS and 4µg/mL sequabrene. Lentiviral aliquots were thawed and 100µL applied to each well in duplicate. Cells were maintained in culture at ° 37 C and 5% CO2 atmospheric condition.

2.6.3 Cloning shRNA into inducible systems

Short-hairpin RNAs (shRNA) targeting Mcl1 were obtained from A/Prof. Ross Dickins (Monash University, Victoria, Australia) and oligonucleotides for shRNAs targeting Cdk9 were ordered using published sequences (Huang et al. 2014). The sequences of Mcl1 and Cdk9 shRNAs are listed in Table 2.6. The Mcl1 constitutive constructs (3µg) and REBIR vector (provided by Mr Sang-Kyu Kim, Johnstone laboratory, PMCC, Victoria, Australia) were digested using CutSmart® (New England Biolabs® Inc., Ipswitch, MA, USA) with EcoRI and XhoI (Promega, Madison, WI, USA) at 37°C for two hours. Digested DNA was labelled and separated using 2% agarose gel with 3µL/mL Midori Green (Bulldog Bio Inc., Portsmouth, NH, USA) then isolated under UV light. DNA was extracted from gel using the Nucleospin® PCR a ligation reaction was performed using 3:1 (insert:vector) in the presence of Clean-up kit (Macherey-Nagel, Bethlehem, PA, USA). Following DNA quantification, T4 ligase (Promega, Madison, WI, USA). Transformation of TOP10F competent bacteria was performed by the addition of 5µL of ligase reaction mixture to 100µL of TOP10F bacteria, placed on ice for 30 minutes then heatshocked at 42°C for 90 seconds in a water bath prior to placing back on ice. Transformed TOP10F bacteria were then washed with 1mL L-broth prior to resuspending in 100µL L-broth which was then applied to L-ampillin-impregnated agar plates. Following 37°C overnight incubation, plates were inspected and independent colonies chosen for amplification as maxi-preparations using 400µL L-broth supplemented 37°C. DNA was extracted using the Nucleospin® PCR Clean-up kit (Macherey- with 267µL ampicillin and placed in flasks on a mechanical shaker overnight at Nagel, Bethlehem, PA, USA) according to manufacturer instructions and DNA quantitation performed prior to storing the constructs at -20°C for future use.

2.6.4 Competitive cell growth/proliferation assays

2.6.4.1 Competitive assays of retrovirus-transduced cells

Competitive proliferation assays were performed by adding 5 x 105 parental cells to 5 x 105 transduced cells in 5mL media cultured in a six-well tissue culture plate. Paired wells either were left untreated or had 1µg/mL doxycycline added to induce expression of short-hairpin RNA. Cells were maintained in usual culture conditions

74 Chapter 2

for proportion of cells expressing the inducible hairpin as represented by inducible and serially passaged. Serial flow cytometric assessment was performed to assess fluorescent protein expression. 2.6.4.2 Competitive assays of lentivirus-transduced cells

OPM2 cells were transduced with lentivirus as described in section 2.6.2.2 above. Triplicate plates were seeded, to allow for serial time-point assessment. Cells ° were maintained in culture at 37 C and 5% CO2 atmospheric condition. At serial time-points, a representative plate for each series of transductions was removed from culture, cells were gently resuspended and 2µL propidium iodide solution added for cell viability assessment. The plates were then analysed for cell viability

Biosciences, San Jose, CA, USA). and GFP-representation with the automated BD FACSVerse flow cytometer (BD

2.7 In vivo experimentation

2.7.1 Experimental animals and housing

All animal experimentation was performed in the animal facility at PMCC (Victoria, Australia). Prospective ethics approval was obtained from the institutional animal experimental ethics committee, permits E472 and E555. All experimentation was purposes. performed within the Australian code for the care and use of animals for scientific

C57BL/6, PTPRCA and NOD-scid IL2Rγnull mice were obtained from the Walter & Eliza Hall Institute breeding facility or the in-house breeding facility at PMCC. Vκ Myc C57BL/6 mice were obtained from the breeding facility at PMCC. Mice were managed by specialist animal technicians and housed in pathogen-free * γnull mice. conditions including use of air-filter boxes for NOD-scid IL2R 2.7.1.1 Preparation of therapeutics for in vivo administration

Therapeutics were freshly prepared prior to administration. The sources, vehicles and schedules of administration for the various compounds are listed in Table 2.7.

75 Chapter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

76 Chapter 2

2.7.1.1.1 A1592668.1

The vehicle for A1592668.1 (Table 2.7) was made without DMSO and was stirred then placed in 42°

C waterbath overnight. A1592668.1 was dissolved in the final was performed and the product was administered. v/v DMSO and added to the vehicle for a final concentration of 0.5mg/mL, vortex

2.7.1.1.2 ABT-199

ABT-199 vehicle was reconstituted by v/v addition of components (Table 2.7) following which ABT-199 was added at 20mg/mL prior to vortex and administration.

2.7.1.1.3 AZ-MCL1

The vehicle for AZ-MCL1 comprised 30% HPBCD supplemented with 1M meglumine according to the following equation:

Vmeglumine (mL)

(mL) = [concentration (20mg/mL)/672.3(g/mole)/1M]*Vfinal meglumine 1M according to the above equation, stirred on a heat pad at 50°C AZ-MCL1 was added to 85% of the final volume of 30% HPBCD supplemented with until granules were no longer visible, then the final 15% of the HPBCD volume was ®, PALL Life Sciences, Port Washington, NY, USA) immediately prior added and stirring continued. The final solution was cleared through a 0.22µm to administration. filter (Acrodisc

2.7.1.1.4 AZD4320

calculated and 80% of this was added to compound and stirred. The solution was AZD4320 was prepared at 2mg/mL. The final volume of 30% HPBCD was until completely dissolved. The solution was then adjusted to pH 4 by addition acidified to pH 2 with addition of 1N methane sulfonic acid and stirring continued of methane sulfonic acid or NaOH as required. The remaining 30% HPBCD was ®, PALL Life Sciences, Port Washington, NY, USA) immediately prior to administration. added and the solution filter-sterilised through a 0.22µm filter (Acrodisc

77 Chapter 2

2.7.1.1.5 Dinaciclib

Dinaciclib was prepared at 4mg/mL in 20% HPBCD vehicle. Following

®, PALL Life Sciences, Port Washington, reconstitution, the product was sonicated until completely dissolved, then filter- NY, USA) immediately prior to administration. sterilised through a 0.22µm filter (Acrodisc

2.7.1.1.6 Fedratinib

The vehicle was prepared by addition of the components (Table 2.7 volume of 100mL sterile distilled H 0. Fedratinib 20mg/mL was added and 2 ) to a final vortex applied to the product until completely dissolved immediately prior to administration.

2.7.1.1.7 Ibrutinib

Ibrutinib was added to vehicle comprising 5% mannitol w/v and 0.5% gelatin w/v. Vortex was applied until the compound completely dissolved immediately prior to administration.

2.7.1.2 Administration of therapeutics

A1592668.1, ABT-199, fedratinib and ibrutinib were administered by oral gavage

PA, USA). Drug concentrations were prepared such that no greater than 200µL using 20G polyurethane flexible feeding tubes (Instech Laboratories, Plymouth, was administered in any single gavage dose. Dinaciclib was administered using a 26G needle (Terumo® Medical, Somerset, NJ, USA) by intraperitoneal injection into the left or right iliac fossae alternating for consecutive doses. AZ-MCL1 and AZD4320 were administered by tail vein injection using a 26G needle (Terumo® Medical, Somerset, NJ, USA) in volumes no greater than 100µL. Following injection, the needle remained in situ for 10 seconds prior to removal in order to minimise extravasation of the compound.

2.7.1.3 Assessment of experimental animals

All animals were regularly assessed independently by animal technicians not directly involved with the experiments. Mice were assessed for signs of general poor health including loss of weight (≥20% baseline), ruffled appearance, hunched posture, rapid breathing, diarrhoea and reduced mobility. Signs of specific tumour 78 Chapter 2 progression were also assessed including bulky lymphadenopathy appearing to cause discomfort, bulky splenomegaly causing hunched posturing, rapid breathing potentially complicating thymic enlargement, frontal bossing of the skull or hind limb paralysis. Any mice deemed to be unwell according to these criteria were humanely euthanized by cervical dislocation.

2.7.2 In vivo experimentation

2.7.2.1 Transplantation of Eµ-Myc lymphoma

Eµ-Myc lymphoma cells were washed and resuspended at 5 x 105 cells/mL in PBS. Non-irradiated C57BL/6 mice were transplanted with 1 x 105 cells in 200µL PBS by intravenous tail vein injection using a 26G needle (Terumo® Medical, Somerset, NJ, USA). Transplanted lymphoma typically engrafted in the lymph nodes, spleen, liver and bone marrow with eventual development of a leukaemic phase of disease as previously described (Shortt et al. 2013).

2.7.2.1.1 Assessment of Full Blood Examination (FBE) and lymphoma burden in blood according to number and GFP representation

Haematological indices from the peripheral blood were assessed following collection of 100µL of blood from the retro-orbital sinus into microcentrifuge tubes containing 10µL EDTA 10mM. From this collection, 25µL was added to 175µL PBS and the full blood examination (FBE) analysis was performed on the CELL-DYN Sapphire Blood Analysis Instrument (Abbott Laboratories, Abbott Park, IL, USA). When GFP-expressing Eµ-Myc lymphoma was used, peripheral blood analysis also included dilution of 5µL blood with 100µL of red cell lysis buffer (150mM NH4Cl,

10mM KHCO3, EDTA 0.1mM and 5% FBS v/v in distilled H20) for two incubations of five minutes each on ice prior to wash and resuspension in 100µL annexin binding II analyser (Franklin Lakes, NJ, USA). buffer and flow cytometric analysis for GFP-representation on the BD FACSCanto™

2.7.2.1.2 In vivo apoptosis assays using Eµ-Myc lymphoma

Additional biomarker studies were performed using in vivo apoptosis assays. Myc lymphoma as described above and left untreated for 12 days to allow development of bulky Briefly, non-irradiated C57BL/6 mice were transplanted with Eµ- was administered at a certain interval prior to mice being euthanased and tissue lymphoma infiltration of the lymph nodes and spleen. A dose of therapy or vehicle

79 Chapter 2

node in 10% neutral buffered formalin for later histological use. Two or more harvested for analysis. This typically involved fixation of a single iliac lymph further lymph nodes were harvested, placed into a 12-well plate and dispersed into a single cell suspension by compression using a 10mL syringe plunger prior to analysis and protein lysate extraction for Western blotting. filtering through a 0.8µm filter in PBS. The specimen was then aliquoted for FACS

2.7.2.2 Transplantation of Vκ*Myc myeloma

C57BL/6 mice were administered 3.3Gy sub-lethal irradiation at two timepoints six hours apart, 24 hours prior to intravenous tail vein transplantation with 1 x 105 Vκ Myc myeloma cells previously harvested from a syngeneic mouse in 200µL PBS by intravenous tail vein injection using a 26G needle (Terumo® Medical, * Somerset, NJ, USA). Mice were housed under standard conditions and bled 34 days later for serum assessment of monoclonal protein production.

2.7.2.3 Transplantation of Burkitt lymphoma cell line

The human BL-41 Burkitt lymphoma cell line containing a bioreporter was cultured in exponential growth phase. Non-irradiated NOD-scid IL2γRnull mice were transplanted with 1 x 106 cells in 200µL PBS by intravenous tail vein injection using a 26G needle (Terumo® Medical, Somerset, NJ, USA).

2.7.3 Histology of in vivo-derived specimens

2.7.3.1 Hematoxylin and eosin stain

were forwarded to the Histology Core Facility (PMCC, Victoria, Australia) where Following fixation in 10% neutral buffered formalin for at least 48 hours, specimens subsequent hematoxylin and eosin staining. Slides were then washed with distilled they underwent processing steps of paraffin fixation, slicing, de-waxing and H2O prior to coverslipping and imaging using the Olympus BX-51 microscope (Olympus, Tokyo, Japan) with SPOT software (Sterling Heights, MI, USA).

2.7.3.2 Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL)

The TUNEL assay was used to assess ex vivo lymph node specimens for cellular apoptosis induction. Formalin-fixed paraffin embedded tissue slices (4µm) 80 Chapter 2 mounted on slides were prepared using the ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Merck KGaA, Darmstadt, Germany) and imaged using the Olympus BX-51 microscope (Olympus, Tokyo, Japan) with SPOT software (Sterling Heights, MI, USA).

2.7.4 Biochemistry assessment

2.7.4.1 Serum protein electrophoresis (SPEP)

Mice were bled by capillary collection from the retro-orbital sinus or from tail tubes without anticoagulant. Blood samples (100µL) were allowed to coagulate vein dissection (Swann-Morton scalpel blade, Sheffield, UK) into microcentrifuge for at least 60 minutes prior to microcentrifugation at 11,000rpm for two minutes in order to separate serum. The serum was then aliquoted into another set of microcentrifugation tubes and stored at -20°C. For analysis, 15µL of serum was applied to independent wells of an applicator and proteins were separated in the diagnostic Biochemistry Laboratory at PMCC (Victoria, Australia) using the Sebia® serum M-spike was performed by diagnostic Biochemistry Laboratory staff using Hydrasys gel electrophoresis system (Sebia, Norcross, GA, USA). Quantification of Phoresis software (Sebia, Norcross, GA, USA).

2.7.4.2 Serum liver, pancreatic and muscle enzyme assessment

Blood was collected by capillary tube venepuncture of the retro-orbital sinus into microcentrifuge tubes and allowed to coagulate for 60 minutes. Samples were centrifuged at 5,000rpm for two minutes in order to separate serum, which was then aliquoted into specimen cups for biochemistry assays using the Abbott ci4100 analyser (Abbott Diagnostics, Abbott Park, IL, USA) in the diagnostic Biochemistry Laboratory at PMCC (Victoria, Australia). Analysis of each specimen was performed in duplicate and the mean value used for subsequent statistical analysis.

2.7.5 In vivo imaging

For in vivo experiments where the lymphoma cells possessed a bioreporter, mice were injected with 10µL/kg of D-luciferin (Thermo Fisher Scientific™, Waltham, 100 Imaging System (PerkinElmer Inc., Boston, MA) and Living Image 3.2 software MA, USA), then anaesthetised with isoflurane and imaged using the Xenogen IVIS (PerkinElmer Inc., Boston, MA). Images were normalized to the same scale in order to allow for comparative assessment between treatment groups.

81 Chapter 2

2.8 Statistical analyses

GraphPad Prism Software, Version 6.0c (La Jolla, CA, USA) with α at 0.05 for Statistical analyses and creation of graphical figures were performed using statistical significance. Combination drug assay synergy was assessed using the Chou- Talalay method (Chou 2006) with Calcusyn software (Biosoft, Cambridge, UK). A combination index >1 was consistent with antagonism.

82 Chapter 3: In vitro and in vivo characterisation of CDK9 inhibition with dinaciclib as an effective therapeutic strategy for MYC-driven B-cell lymphoma

83 Chapter 3

3.1 Introduction

MYC-dysregulated B-cell lymphoma encompasses a number of diagnoses as and Lymphoid Tissues (Swerdlow et al. 2008). In contrast to high cure rates classified according to the 2008 WHO Classification of Tumours of Haematopoietic achieved with immunochemotherapy for Burkitt lymphoma, MYC-dysregulation appears to confer poor prognosis to DLBCL (Savage et al. 2009; Barrans et al. 2010; Perry et al. 2014; Copie-Bergman et al. 2015). Furthermore, elderly and frail patients are often unable to tolerate the intensive chemotherapy regimens required to induce remission and possible cure in these diseases. There remains an unmet clinical need for further effective therapeutic strategies for the treatment of aggressive MYC-dysregulated lymphoma.

As a transcription factor and master transcriptional regulator, MYC is involved with transcription of a myriad of target genes (Li et al. 2003). However, contrasting evidence exists as to whether MYC is directly involved in recruitment of transcriptional complex components, or whether it is involved in pause-release of already bound Pol II to facilitate effective transcriptional elongation (Kanazawa et al. 2003; Marshall et al. 1996; Gargano et al. 2007; Cowling and Cole 2007; Rahl et al. 2010). In diseases driven through MYC-dysregulation, a state of global transcriptional upregulation exists (Dave et al. 2006), potentially rendering components of the transcriptional elongation complex as targets through which to uncouple the complex and reduce the dysregulated transcription though which tumorigenesis is maintained.

Pol II is fully activated by phosphorylation of serine residues within heptapeptide which is essential for effective transcriptional elongation to occur (Marshall et al. repeats in the CTD sequentially at position five then position two, the latter of 1996). Serine two CTD phosphorylation is predominantly performed by CDK9 following its activation upon binding cyclin T1, to form the positive transcription elongation factor, P-TEFb (Marshall and Price 1992; 1995) (Figure 3.1).

results for the treatment of the indolent B-cell lymphoproliferative disorder CLL Previous clinical trials of the pan-CDK inhibitor flavopiridol yielded disappointing with only a minority of patients achieving partial responses and few complete responses (Byrd et al. 2005; 2007; Lin et al. 2009; Phelps et al. 2009). Concurrent associated reduction in expression of the anti-apoptotic protein MCL-1 reported preclinical evidence linked flavopiridol to reduced activation of Pol II with (Gojo et al. 2013; Chen et al. 2005b). However, recent success with the use of

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Dinaciclib

PTEFb Cyclin T1 CDK9 MYC YSPTSPS/YSPTSPS RNA polymerase II

Transcription

Mcl-1

Figure 3.1: CDK9 regulates full activation of Pol II-mediated transcription of critical MYC targets including MCL-1

Schematic representation of the proposed oncogenic pathway demonstrating MYC binding of cyclin T1 to recruit P-TEFb to MYC transcriptional target sites. CDK9 phosphorylates RNA Polymerase II (Pol II), subunit B1 at serine residues at position two within heptapeptide repeats in the carboxy-terminal domain, fully activating Pol II to commence effective transcriptional elongation. MCL-1 is depicted as a critical target of MYC-addicted transcription. Dinaciclib is represented as an inhibitor of CDK9. P denotes phosphorylation.

85 Chapter 3 the BCL-2 inhibitor, navitoclax (ABT-199) (Roberts et al. 2016), suggests that CLL dependency upon all anti-apoptotic BCL-2 family members, in particular MCL-1. may have a particular oncogenic dependency upon BCL-2, rather than non-specific

CDK inhibitor with low nanomolar activity against CDK1, 2, 5 and 9 (Parry et al. Dinaciclib (Merck, Boston, MA) was developed as a more specific and potent in CLL, acute myelogenous leukaemia, multiple myeloma and a range of solid 2010). The favourable inhibitory profile of dinaciclib led to clinical development organ malignancies (Gojo et al. 2013; Nemunaitis et al. 2013; Flynn et al. 2015; Kumar et al. 2014). Despite evident clinical activity, development of dinaciclib was largely halted due to market competition with the success of navitoclax targeting BCL-2 and ibrutinib targeting Bruton tyrosine kinase (BTK) for treatment of CLL (Byrd et al. 2013; Roberts et al. 2015). We hypothesised that the transcriptionally addicted state of MYC-dysregulated lymphoid malignancy may confer an oncogenic dependency upon MYC-transcriptional targets including MCL-1, which provided the rationale for studies of CDK9 inhibition with dinaciclib in MYC-driven lymphoma models described in this chapter. Herein, we use in vitro and in vivo studies to show

The chapter comprises preliminary unpublished experimental work followed by a significant activity of dinaciclib, including occasional observed curative responses. peer-reviewed publication (Gregory et al. 2015).

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3.2 Results

3.2.1 Dinaciclib treatment potently induces apoptosis of Eµ-Myc lymphoma and human IG-cMYC-translocated lymphoma in vitro

Cell-free kinase inhibitory assays have shown dinaciclib to have an IC50 of 1, 1, 3 and 4 nmol/L for CDK2, CDK5, CDK1 and CDK9, respectively (Parry et al. 2010). In order to assess the effect of dinaciclib on Eµ-Myc lymphoma cell viability, a representative Eµ-Myc cell line was cultured in vitro with increasing concentrations of dinaciclib for 24 hours prior to assessment for cell death according to annexin-V Figure 3.2a,b). Further analyses demonstrated that p53-competent (#30 Bmf-/-, #20 Bim-/-, #6066NRASQ61K, #4242) and p53-null / PI staining by flow cytometry ( (#3391) lymphoma cell lines were sensitive to dinaciclib-induced cell death at low nanomolar concentrations approaching those quoted for ‘on-target’ kinase inhibition (Figure 3.2c, Gregory et al 2015 Figure 1a), whereas forced overexpression of the anti-apoptotic proteins Bcl-2 or Mcl-1 driven by a retroviral promoter (#4242tmBcl-2, #4242tmMcl-1) was protective against dinaciclib- induced cell death (Figure 3.2d, Gregory et al 2015 Figure 1g). Dinaciclib was similarly potent against a panel of human IG-cMYC-translocated Burkitt lymphoma and multiple myeloma cell lines upon 48 hour incubation and analysis according to annexin-V / PI-positivity (Figure 3.2e). This suggests that dinaciclib is able to induce cell-autonomous apoptosis upon incubation at low nanomolar concentrations targeting CDK9.

When Eµ-Myc lymphoma overexpressing Bcl-2 (#4242tmBcl-2) was exposed to dinaciclib for 24 hours, cell cycle analysis according to Nicoletti protocol also showed a population of cells with fragmented DNA at low nanomolar concentrations (Figure 3.3), corresponding with apoptotic cell death. This proportion of cells was attenuated when compared to the amount of cell death seen with the apoptosis assays of parental cells, consistent with overexpression of anti-apoptotic Bcl-2 in

S-phase were also observed in these apoptosis-protected cells at low nanomolar this instance. Of interest, a significant increase in G2/M-phase and reduction in concentrations. In conjunction with the apoptosis assay results (Figure 3.2), these when this is suppressed through overexpression of anti-apoptotic Bcl-2, the cells findings indicate that the primary response to dinaciclib is apoptosis, however, still respond biologically through cell cycle arrest.

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Figure 3.2: Dinaciclib induces apoptosis of Eµ-Myc and human IG-cMYC- translocated lymphoma in vitro

(a) Myc lymphoma cell line (#6066) was cultured for 24 hours in vitro with varying concentrations of dinaciclib (4 – 16nM) or An exponentially growing Eμ- DMSO vehicle as represented by 0nM. Flow cytometric analysis was performed at 24 hours. Representative gating strategy with forward scatter (FSC) and side scatter (SSC) showing a shift from viable to non-viable cells with increasing concentrations of dinaciclib. (b) Expression of annexin-V and propidium iodide (PI) uptake of lymphoma cells from the same experiment (#6066). (c) Bar graphs depict the mean proportion of non-viable lymphoma cells according to annexin-V / PI uptake for each assessed concentration of dinaciclib or DMSO vehicle (0nM) for Myc lymphoma cell lines. (d) Myc lymphoma cell line #4242 was stably transduced with an expression vector MSCV-Bcl-2-GFP and assessed representative Eμ- Eμ- for proportion of apoptotic cells according to annexin-V / PI uptake following 24 hour incubation with represented concentrations of dinaciclib. (e) Human IG- cMYC translocated Burkitt lymphoma cell lines (grey bars) and multiple myeloma cell lines (white bars) were cultured with dinaciclib in vitro for 48 hours prior to assessment of cell viability according to annexin-V / PI uptake.

Data representative of mean +/- standard error of the mean of non-viable cells for three independent experiments.

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a DMSO (0nM) 4nM 8nM 16nM SSC FSC b PI

Annexin-V

c # -/- #6066 NRASQ61K 30 Bmf #20 Bim-/- I I 100 100 I 100 / P / P / P n n n 50 50 50 nn ex i nn ex i nn ex i A

A A

% % 0 0 % 6 0 4 8 0 0 4 8 6 6 0 4 8 1 1 1 Dinaciclib [nM] Dinaciclib [nM] Dinaciclib [nM] d e #4242tmBcl-2 BL-41 Ramos NamalwaOPM2 H929 I I 100 100 / P / P n n 50 50 nn ex i nn ex i A A

% % 0 0 6 6 6 6 6 0 4 8 0 4 8 0 4 8 0 4 8 0 4 8 6 0 4 8 1 1 1 1 1 1 Dinaciclib [nM] Dinaciclib [nM]

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a DMSO (0nM) 4nM 16nM 64nM

PI

b #4242tmBcl-2 G2-M 100 S G1 SubG1 ce ll s l

a 50 To t

%

0 6 2 3 0 4 8 1 3 6 12 5 Dinaciclib [nM]

Figure 3.3: Dinaciclib treatment is associated with cytostasis and apoptosis of Eµ-Myc lymphoma in vitro

(a) Myc lymphoma overexpressing Bcl-2 (#4242tmBcl-2) was incubated with various concentrations of dinaciclib or DMSO vehicle for 24 hours prior to Eμ- plots depict nuclear DNA content as assessed by PI uptake. (b) Cigar plots for the preparation of cells using the Nicoletti protocol. Representative flow cytometry same experiment showing proportion of cells in each phase of the cell cycle for representative dinaciclib concentrations.

Data representative of mean +/- standard error of the mean for three independent experiments.

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3.2.2 Dinaciclib treatment of Eµ-Myc lymphoma leads to CDK9 inhibition of RNA polymerase II activation and reduced Mcl-1 protein expression

In order to demonstrate CDK9 inhibition by dinaciclib, apoptosis-protected Eµ- Myc lymphoma cells overexpressing Bcl-2 (#4242tmBcl-2) were exposed to varying concentrations of dinaciclib for one hour prior to Western blot assessment of RNA polymerase II, subunit B1 CTD phosphorylation (pRpb1). A clear reduction of pRpb1(Ser2/5) was observed at low nanomolar concentrations of dinaciclib following one hour incubation and was also observed upon at least 24 hours of incubation (Figure 3.4a,b). Incubation with dinaciclib for 24 hours was also associated with reduced expression of Mcl-1 protein, with no discernible effect on Bcl-xL, Bim or Bmf protein levels (Figure 3.4c). Repeat experiments with non- transduced Eµ-Myc in the CDK9 target pRpb1(Ser2), but not the CDK7 target pRpb1(Ser5) (Gregory lymphoma also showed specificity of dinaciclib for reduction et al 2015 Figure 1e).

When the same experiment was performed using non-transduced #4242 lymphoma cells at 6 hours incubation with dinaciclib, a reduction in pRpb1(Ser2/5) and Mcl-1 was again observed (Figure 3.5a). Notably, while the 20nM concentration lower concentration of 10nM a preferential reduction in the longer isoform was was observed to significantly reduce expression of both isoforms of Mcl-1, at the shown previously that in contrast to the pro-apoptotic short splice variants of Mcl- observed. This finding is not discordant with the apoptosis assays, as it has been 1, the long isoform functions in an anti-apoptotic manner (Kozopas et al. 1993; Bae et al. 2000; Kim et al. 2009; Ménoret et al. 2010; Thomas et al. 2010). Hence, select reduction of this long isoform is consistent with observed apoptosis induction. When the experiment was repeated using the #4242 lymphoma transduced with a construct for forced overexpression of Mcl-1 driven by a retroviral promoter (#4242tmMcl-1), dinaciclib-mediated repression of Mcl-1 levels was completely abrogated despite reduction in pRpb1(Ser2/5) (Figure 3.5b). This suite of correlate with inhibition of Pol II activation and are associated with acute experiments confirms that low nanomolar apoptotic concentrations of dinaciclib reductions in Mcl-1 protein expression.

3.2.3 Dinaciclib therapy is associated with CDK9 inhibitory activity and prolonged survival of tumour-bearing mice in vivo

In order to validate dinaciclib as a therapeutic option for MYC-driven lymphoma,

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Figure 3.4: Dinaciclib treatment is associated with reduced Pol II activation and Mcl-1 protein expression in apoptosis- protected lymphoma cells

(a) Myc lymphoma cells overexpressing Bcl-2 (#4242tmBcl-2) were cultured in the presence of varying concentrations of Apoptosis-protected Eμ- dinaciclib, DMSO or untreated for one hour or 24 hours prior to Western blotting. polyacrylamide gel using SDS-PAGE prior to immunoblotting for RNA polymerase Protein extracted from whole cell lysates (10μg) was separated on gradient II, subunit B1 phosphorylation at Serine 2/5 (pRpb1Ser2/5) and HSP90 loading control. (b) Myc lymphoma cells (#4242tmBcl-2) were cultured in the presence of 10nM dinaciclib or DMSO vehicle for one hour prior to Western blotting. Eμ- polyacrylamide gel using SDS-PAGE prior to immunoblotting for pRpb1Ser2/5 and Protein extracted from whole cell lysates (10μg) was separated on gradient HSP90 loading control. (c) Myc lymphoma cells (#4242tmBcl-2) were cultured in the presence of dinaciclib 10nM or DMSO vehicle control for 24 hours prior to Eμ- on gradient polyacrylamide gel using SDS-PAGE prior to immunoblotting for Mcl- Western blotting. Protein extracted from whole cell lysates (10μg) was separated 1, Bcl-xL, Bmf, Bim and HSP90 loading controls.

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a 1 h 24 h Dinaciclib - - + + + + - - + 50nM 25nM 100nM Untreated DMSO 12.5nM Untreated DMSO 10nM

pRpb1Ser2/5 250 kDa

HSP90 90 kDa b c 1 h 24 h DMSO Dinaciclib DMSO Dinaciclib

Ser2/5 pRpb1 250 kDa Mcl-1 40 kDa

HSP90 90 kDa HSP90 90 kDa

Bcl-xL 30 kDa

HSP90 90 kDa

35 kDa Bmf 25 kDa

23 kDa Bim 15 kDa

HSP90 90 kDa

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Figure 3.5: Dinaciclib treatment is associated with reduced Pol II activation and Mcl-1 protein expression in wild-type lymphoma cells

(a) Myc #4242 lymphoma cells were cultured in the presence of displayed concentrations of dinaciclib or DMSO for six hours prior to Western blotting. Eμ- polyacrylamide gel using SDS-PAGE prior to immunoblotting for pRpb1Ser2/5, Mcl- Protein extracted from whole cell lysates (10μg) was separated on gradient 1 and HSP90 loading control. (b) Myc lymphoma # Myc lymphoma #4242 cells transduced with an expression vector MSCV-Mcl-1-GFP Eμ- 4242 cells and Eμ- (#4242tmMcl-1) were cultured in the presence of dinaciclib or DMSO for six hours separated on gradient polyacrylamide gel using SDS-PAGE prior to immunoblotting prior to Western blotting. Protein extracted from whole cell lysates (10μg) was for pRpb1Ser2/5, Mcl-1 and HSP90 loading control.

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a #4242 DMSO Dinaciclib 10nM Dinaciclib 20nM

pRpb1Ser2/5 250 kDa

Mcl-1 40 kDa

HSP90 90 kDa

b #4242 #4242tmMcl-1 DMSO Dinaciclib Untreated DMSO Dinaciclib

pRpb1Ser2/5 250 kDa

Mcl-1 40 kDa

HSP90 90 kDa

95 Chapter 3 a series of pre-clinical in vivo experiments were next performed to assess whether the activity observed in vitro translates to in vivo work in the Johnstone laboratory performed by Ms Adele Baker showed in vivo efficacy. Previous experimental tolerability of dinaciclib in C57BL/6 mice (Baker et al. 2016), consistent with studies performed by our commercial pharmaceutical collaborators (Merck) indicating a maximum tolerated dose of 40mg/kg administered every three days by intraperitoneal injection (data not shown).

lead to the same signalling changes in tumour bearing mice as were observed by The first experiments were aimed to determine whether dinaciclib treatment would Western blot in vitro (Figures 3.4-3.5). Syngeneic mice were transplanted with Eµ-Myc lymphoma and left untreated to establish bulky nodal disease. Following a single dose of dinaciclib or vehicle [20% hydroxypropylbetacyclodetran (HPBCD)], lymph node-derived cell lysates were obtained one or four hours later Figure 3.6), demonstrating the same effect of dinaciclib in vivo as to that observed in and confirmed Pol II inhibition and reduction of Mcl-1 protein expression ( vitro histology also indicated rapid and potent apoptosis induction (Gregory et al 2015 . Furthermore, independent readouts of apoptosis by flow cytometry and Supplementary Figure S4).

Having shown activity of dinaciclib in vivo, the next experiments were performed

Myc lymphoma. Syngeneic mice were transplanted with Eµ-Myc lymphoma then to assess whether dinaciclib could provide a survival benefit to mice bearing Eµ- received six weeks of therapy with dinaciclib or vehicle (Figure 3.7a). Dinaciclib therapy was mostly well tolerated with occasional gastro-intestinal toxicity in weight loss was observed in either the vehicle or dinaciclib treatment arms (data the form of ileus causing distended stomach and proximal bowel. No significant not shown). Disease biomarker studies using bioluminescence imaging and assessment of number of lymphoma cells in leukaemic phase in the peripheral Figure 3.7b,c blood showed dinaciclib to significantly abrogate disease progression ( advantage across a suite of different tumour clones that were assessed including ). This delay in disease progression conferred a significant survival occasional curative responses in sensitive clones (Figure 3.7d,e, Gregory et al 2015 Figure 2a-d). These responses are far superior to other targeted therapies assessed for treatment of Eµ-Myc lymphoma in vivo, including ibrutinib to target Bruton tyrosine kinase (Figure 3.8a) and fedratinib to target JAK2 signalling (Figure 3.8b).

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a Eµ-Myc 1x105 cells

Day 12

20% HPBCD vehicle vs. Dinaciclib 30mg/kg by intraperitoneal injection

b ehicle (4h) V Untreated Dinaciclib (4h) Dinaciclib (1h)

pRpb1Ser2/5 250 kDa

Mcl-1 40 kDa

HSP90 90 kDa

Figure 3.6: Dinaciclib therapy is associated with reduced Pol II activation and Mcl-1 protein expression in vivo

(a) Non-irradiated C57BL/6 recipient mice were transplanted by tail vein injection with 1 x 105 -Myc #4242 lymphoma cells harvested from lymph nodes of a syngeneic mouse. After allowing 12 days to establish bulky nodal disease, mice Eμ received a single dose of vehicle 20% hydroxypropyl-beta-cyclodextrin (HPBCD) were harvested from independent mice and prepared as a single cell suspension or dinaciclib 30mg/kg, prior to sacrifice at one or four hours later. Lymph nodes from which protein was extracted from whole cell lysates. (b) Protein extracted using SDS-PAGE prior to immunoblotting for pRpb1Ser2/5, Mcl-1 and HSP90 loading from whole cell lysates (10μg) was separated on gradient polyacrylamide gel control. Each lane represents protein from cell lysate derived from lymph nodes of a single mouse.

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Figure 3.7: Dinaciclib therapy is associated with biomarkers of disease response and prolongation of overall survival in vivo

(a) Schematic representation of dinaciclib therapy studies to assess for overall survival. Non-irradiated C57BL/6 recipient mice were transplanted by tail vein injection with 1 x 105 -Myc lymphoma cells harvested from lymph nodes of a syngeneic mouse. Therapy was commenced three days later with 20% HPBCD Eμ vehicle or dinaciclib 30mg/kg, administered twice weekly by intraperitoneal injection for a total of six weeks. Ethical endpoints of illness were independently assessed by animal technicians not involved with the experiment. (b) Dinaciclib Myc lymphoma #6066 transduced with a bioluminescence reporter gene. Disease burden for each treatment group therapy experiment of mice transplanted with Eμ- at day seven post transplantation is shown. (c) Mice from the same experiment were bled at day seven and assessed by full blood examination for absolute number of lymphocytes in peripheral blood (lymphoblasts in leukaemic phase). (d) Myc lymphoma #6066 experiment. Median survival for vehicle-treated mice (n=9) was 10 days and for Kaplan-Meier survival curve of mice from the same Eμ- dinaciclib-treated mice (n=9) was 21 days (p<0.0001, Log-rank test). (e) Kaplan- Myc lymphoma #107. Median survival for vehicle-treated mice (n=6) was 13 days and for dinaciclib-treated mice Meier survival curve of mice transplanted with Eμ- (n=6) was 39 days (p=0.001, Log-rank test).

Grey shading denotes period of therapy administration. Error bars denote mean p<0.001 comparing vehicle and dinaciclib- treated groups (unpaired student’s t-test). +/- standard error of the mean. ***

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a Eµ-Myc 1x105 cells

Day 3 Day 42

20% HPBCD vehicle vs. Dinaciclib 30mg/kg twice weekly by intraperitoneal injection

b c #6066 ***

/L ) 20 9 Vehicle x1 0 (

es 10

Dinaciclib ym pho cy t

L 0 e Vehicl Dinaciclib

d e # #6066 107 100 100 Dinaciclib Dinaciclib va l va l Vehicle Vehicle rv i rv i

u 50

u 50 S

S

% % 0 0 0 20 40 60 0 20 40 60 Days post transplantation Days post transplantation

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Figure 3.8: Comparative overall survival conferred by other targeted therapies against Eµ-Myc lymphoma in vivo

(a) Non-irradiated C57BL/6 recipient mice were transplanted by tail vein injection with 1 x 105 Myc #6066 lymphoma cells harvested from lymph nodes of a syngeneic mouse. Therapy was commenced three days later with 5% mannitol Eμ- vehicle or ibrutinib 50mg/kg, administered daily by oral gavage for a total of six weeks. Kaplan-Meier overall survival (OS) curve shows comparative survival for vehicle and ibrutinib-treated mice. Median OS was 8 days for vehicle treated group (n=10) and 7.5 days for ibrutinib-treated group (n=10), p=0.09 (Log-rank test). (b) Non-irradiated C57BL/6 recipient mice were transplanted by tail vein injection with 1 x 105 Myc #4242 lymphoma cells harvested from lymph nodes of a syngeneic mouse. Therapy was commenced three days later with 5% mannitol Eμ- vehicle or ibrutinib 50mg/kg, administered daily by oral gavage for a total of six weeks. Kaplan-Meier overall survival curve shows comparative survival for vehicle and ibrutinib-treated mice. Median OS was 12 days for vehicle treated group (n=10) and 8 days for ibrutinib-treated group (n=10), p=0.0003 (Log-rank test). (c) Non- irradiated C57BL/6 recipient mice were transplanted by tail vein injection with 1 x 105 Myc #6066 lymphoma cells harvested from lymph nodes of a syngeneic mouse. Therapy was commenced three days later with 0.5% methylcellulose Eμ- vehicle or fedratinib 100mg/kg, administered twice daily by oral gavage for a total of six weeks. Kaplan-Meier overall survival curve shows comparative survival for vehicle and fedratinib-treated mice. Median OS was 8 days for vehicle treated group (n=10) and 10 days for fedratinib-treated group (n=10), p<0.0001 (Log- rank test). (d) Non-irradiated C57BL/6 recipient mice were transplanted by tail vein injection with 1 x 105 Myc #4242 lymphoma cells harvested from lymph nodes of a syngeneic mouse. Therapy was commenced three days later with 0.5% Eμ- methylcellulose vehicle or fedratinib 100mg/kg, administered twice daily by oral gavage for a total of six weeks. Kaplan-Meier overall survival curve shows comparative survival for vehicle and fedratinib-treated mice. Median OS was 12 days for vehicle treated group (n=5) and 16.5 days for fedratinib-treated group (n=10), p=0.0002 (Log-rank test).

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a b #6066 #4242

100 Vehicle 100 Vehicle va l Ibrutinib va l Ibrutinib rv i 50 rv i u 50 u S S

% % 0 0 0 20 40 60 0 20 40 60 Days post transplantation Days post transplantation

c d #6066 #4242

100 Vehicle 100 Vehicle va l va l Fedratinib Fedratinib rv i rv i 50

50 u u S S

% % 0 0 0 20 40 60 0 20 40 60 Days post transplantation Days post transplantation

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3.2.4 CDK9 inhibition by dinaciclib potently suppresses Mcl-1 to induce durable apoptotic responses in aggressive MYC-driven B-cell lymphoma in vivo

Gregory GP, Hogg SJ, Kats LM, Vidaks E, Baker AJ, Gilan O, Lefebure M, Martin BP, Dawson MA, Johnstone RW, Shortt J. Leukemia 2015; 29:1437-1441.

This peer-reviewed publication contains further in vitro and in vivo studies to

Aside from similar experiments to those described in the preliminary studies characterise the efficacy of dinaciclib in MYC-driven models of B-cell lymphoma. mentioned above, further mechanistic interrogation of dinaciclib’s action are shown. These studies include chromatin immunoprecipitation PCR to demonstrate CDK9 activity at the Mcl-1 locus, and the associated effects of dinaciclib-mediated CDK9 repression of Pol II activation on anti-apoptotic mRNA expression. Furthermore, not pRpb1(Ser5) is shown. specificity of dinaciclib for preferential dephosphorylation of pRpb1(Ser2), and

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Letters to the Editor 1437 either as false negatives of the PCR or as false positives of MFC. We REFERENCES can thus conclude that the junction region of the IgH rearrange- 1 Nowell PC. The clonal evolution of tumor cell populations.Science1976; 194: 23–28. ment in MM is stable and can be used as a target for MRD 2 Hallek M, Bergsagel PL, Anderson KC. Multiple myeloma: increasing evidence for a assessment by ASO RQ-PCR and more, also by deep-sequencing multistep transformation process. Blood 1998; 91: 3–21. methods, as it constantly identifies the myeloma cells responsible 3 López-Corral L, Sarasquete ME, Beà S, García-Sanz R, Mateos MV, Corchete LAet al. for relapse.15 SNP-based mapping arrays reveal high genomic complexity in monoclonal In conclusion, our results show that, in the dominant myeloma gammopathies, from MGUS to myeloma status. 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OPEN CDK9 inhibition by dinaciclib potently suppresses Mcl-1 to induce durable apoptotic responses in aggressive MYC-driven B-cell lymphoma in vivo

Leukemia(2015) 29, 1437–1441; doi:10.1038/leu.2015.10 recruits transcription complexes containing RNA polymerase II (Pol II) to facilitate e ective transcriptional elongation of MYC gene targets.3 Pol II is fully activated by phosphorylation of a MYC dysregulation confers a poor prognosis to di use large B-cell critical serine residue at position 2 within heptapeptide repeats in lymphoma (DLBCL), and e ective therapeutic strategies are the carboxy-terminal domain (CTD), a function performed by the lacking in relapsed/refractory DLBCL, Burkitt lymphoma and positive transcription elongation factorb(P-TEFb; comprising intermediate forms.1,2 As a master transcriptional regulator, MYC CDK9 and cyclin T1).4 It has been shown that MYC binds and

Accepted article preview online 12 January 2015; advance online publication, 3 February 2015

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Letters to the Editor 1438 a #4242 #3391 bcBL-41 Ramos Bcl-2 Mcl-1 p53 null NS * 100 100 1.5 50

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(DMSO) 0.5 % Annexin / PI % Annexin % Annexin / PI % Annexin Relative mRNA level Relative

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Tubulin 52 kDa c-Myc 70 kDa

Mcl-1 40 kDa Bcl-xL 30 kDa

Bcl-2 26 kDa HSP90 90 kDa

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Figure 1. Dinaciclib potently induces apoptosis of murine Eμ-Myc and human IG-cMYC-translocated lymphomas with rapid and selective suppression of Mcl-1 transcription and protein levels. (a) Wild-type p53 (#4242) and p53-null (#3391) Eμ-Myc lymphomas were cultured in vitro with dimethylsulfoxide (DMSO) vehicle control or dinaciclib for 24 h and then analyzed using flow cytometric analysis for annexin-V/ propidium iodide (PI) positivity. (b) Human IG-cMYC-translocated BL-41 and Ramos cell lines were cultured in vitro with DMSO or dinaciclib for 48 h before the analysis of annexin-V/PI positivity using flow cytometry. (c) Mcl-1 and Bcl-2 mRNA expression in lymphoma #4242 following 3-h in vitro treatment with DMSO or 20 nM dinaciclib. Transcript levels are represented as fold change compared with DMSO. NS, not significant; *Po0.0001. (d) Chromatin immunoprecipitation-PCR of Eμ-Myc lymphoma #4242 cells showing binding of phospho-RNA Pol II CTD serine 2 (pRpb1 Ser2) at the Mcl-1 locus. Error bars denote the s.e.m. from three independent primer sets across the Mcl-1 locus. (e)Eμ-Myc # lymphoma 4242 was cultured in vitro for 3-h untreated or in the presence of DMSO or 20 nM dinaciclib before the preparation of lysates and Ser2 Ser5 Ser2/5 immunoblotting for phospho-RNA Pol II CTD (pRpb1 , pRpb1 and pRpb1 ), total Mcl-1, Bcl-2, Bcl-xL, c-Myc and HSP90 loading control. (f) Human IG-cMYC-translocated BL-41 and Ramos cell lines were cultured in vitro for 3 h in the presence of DMSO or 20 nM dinaciclib before the preparation of lysates and immunoblotting for total Mcl-1, Bcl-2, Bcl-xL, c-Myc, Tubulin and HSP90 loading controls. (g)Eμ-Myc lymphoma #4242 was transduced with murine stem cell virus expressing empty vector control or Mcl-1 and then cultured in vitro with dinaciclib for 24 h before flow cytometric analysis for annexin-V/PI positivity. **Po0.01 comparing treatments at 16 nM concentration. All graphs represent the mean ± s.e.m (error bars) for three or more independent experiments.

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Letters to the Editor 1439 #4242 #3391 #106 p53 null dn p53

100 Dinaciclib 100 Dinaciclib 100 Dinaciclib Vehicle Vehicle Vehicle

50 50 50 Percent survival Percent Percent survival Percent Percent survival Percent 0 0 0 0 40 80 0 40 80 0 40 80 Days post transplantation Days post transplantation Days post transplantation

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50 Mcl-1 40 kDa Percent survival Percent 0 0 40 80 Days post transplantation HSP90 90 kDa

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BL-41 100 Dinaciclib Vehicle Day 7

50 Percent survival Percent 0 0 10 20 30 Day 14 Days post transplantation

Figure 2. Dinaciclib therapy prolongs the survival of mice bearing Eμ-Myc and human IG-cMYC-translocated lymphomas. (a–d) Kaplan–Meier survival curves representing cohorts of C57Bl/6 mice transplanted with representative Eμ-Myc lymphomas 3 days before the therapy commencement with 20% hydroxypropyl-beta-cyclodextran (HPBCD) vehicle or 30 mg/kg dinaciclib by intraperitoneal injection twice weekly. Gray shading denotes the period of therapy. dn, dominant negative; Po0.0001 for each experiment. The median survival for vehicle- and dinaciclib-treated mice were 12 days and not reached (#4242), 16 and 48 days (#3391), 18 and 66 days (#106) and 13 and 18 days (#4242tMcl-1), respectively. (e) Lymph nodes were harvested from cohorts of C57Bl/6 mice 1 or 4 h following a single dose of dinaciclib or 20% HPBCD, 12 days following transplantation with Eμ-Myc lymphoma #4242. Protein lysates were then prepared and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis before immunoblotting for the indicated targets. Each lane represents protein lysate from the lymph nodes of an individual mouse. (f) Bioluminescence imaging of NOD-scid IL2Rγnull mice transplanted with human IG-cMYC-translocated BL-41.luc2 lymphoma 3 days before the commencement of the therapy with 20% HPBCD vehicle or 30 mg/kg dinaciclib by intraperitoneal injection twice weekly. Mice were imaged at 7 and 14 days post transplantation. (g) Overall survival of the mice from the experiment is described in f. Gray shading denotes the period of therapy. The median survival for vehicle and dinaciclib-treated mice were 19 and 26 days, respectively (Po0.001). recruits P-TEFb to its targets as a means to activate Pol II.3,5,6 More transcription of critical MYC-regulated oncogenic effector proteins. recently, CDK9-mediated transcriptional elongation was reported Here we describe durable in vivo responses to dinaciclib in as essential for tumor maintenance in a genetically defined aggressive MYC-driven lymphoma, mediated by downregulation MYC-driven model of hepatocellular carcinoma.7 Thus, CDK9 of Pol II-mediated Mcl-1 transcription. dependence may represent a druggable vulnerability in lymphomas Dinaciclib has 50% kinase inhibitory concentrations of 1, 1, 3 8 with dysregulated MYC expression. and 4 nM for CDK2, CDK5, CDK1 and CDK9, respectively. Dinaciclib Dinaciclib (Merck, Boston, MA, USA) is a novel CDK inhibitor that potently killed Eμ-Myc and human IG-cMYC-translocated cell lines has reached phase 1b/2 of clinical trials for a range of solid-organ independent of p53 function, but not untransformed murine malignancies, as well as for myeloma and chronic lymphocytic fibroblast cells, at low nanomolar concentrations approximating leukemia.8 We hypothesized that CDK9 inhibition by dinaciclib those observed for kinase inhibition (Figures 1a and b, would represent a rational pharmacologic approach to target the Supplementary Figure S1).

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Letters to the Editor 1440 As Bcl-2 and Mcl-1 have been implicated as important apoptotic ACKNOWLEDGEMENTS 9,10 regulators in Eμ-Myc lymphomas, we assessed the effects of Researchers are supported by funding from the Leukaemia Foundation of Australia dinaciclib on these proteins. We hypothesized that CDK9 (GPG, SJH, ML, MAD), the Royal Australasian College of Physicians (GPG) and the inhibition with dinaciclib would target Mcl-1 transcription, as has Arrow Bone Marrow Transplant Foundation (AB, ML). LMK is supported by a CJ Martin been observed with other CDK inhibitors in myeloma and mantle Fellowship (NHMRC), MAD is supported by VESKI Innovation and Herman Clinical cell lymphoma.11,12 Eμ-Myc and human IG-cMYC-translocated cell Research Fellowships, JS is supported by funding from the Eva and Les Erdi/ lines were treated with dinaciclib or dimethylsulfoxide control and Snowdome Foundation Victorian Cancer Agency Fellowship, RWJ is a Principal Research Fellow of the National Health and Medical Research Council of Australia interrogated using the quantitative PCR analysis for the effect on (NHMRC) and is supported by NHMRC Program and Project Grants, the Cancer Mcl-1 and Bcl-2 mRNA. Dinaciclib treatment was associated with a fi fi Council Victoria and the Victorian Cancer Agency. Dinaciclib was provided by signi cant reduction in Mcl-1 mRNA, with no signi cant effect on Merck Research Laboratories (Boston, MA, USA) and α-Amanitin was kindly Bcl-2 transcript levels (Figure 1c, Supplementary Figure S2). provided by Ms Christina Woelwer. We thank Mr Don Cameron for technical advice Chromatin immunoprecipitation-PCR was used to show the regarding α-Amanitin experiments. The TRMPVIR vector was kindly provided by binding of phosphorylated Pol II, subunit B1 carboxy-terminal Dr Johannes Zuber. domain (CTD) serine 2 (pRpb1 Ser2) as a marker of CDK9 activity at the Mcl-1 locus in a representative Eμ-Myc lymphoma cell line GP Gregory1,2,3, SJ Hogg1,2, LM Kats1,2, E Vidacs1,2, AJ Baker1,2, (Figure 1d). These findings support the hypothesis that dinaciclib O Gilan2,4, M Lefebure1,2, BP Martin1,2, MA Dawson2,4, transcriptionally downregulates Mcl-1. RW Johnstone1,2,6 and J Shortt1,2,3,5,6 We next examined Mcl-1 expression in Eμ-Myc and human IG- 1Gene Regulation Laboratory, Research Division, Peter MacCallum cMYC-translocated lymphoma cell lysates following the treatment Cancer Centre, Melbourne, Victoria, Australia; with dinaciclib or vehicle. On-target CDK9 inhibition by dinaciclib 2Sir Peter MacCallum Department of Oncology, The University of was confirmed through inhibition of pRpb1 Ser2 at concentrations Melbourne, Parkville, Victoria, Australia; corresponding to apoptosis induction in Eμ-Myc cells (Figure 1e). 3Monash Haematology, Monash Health, Clayton, Victoria, Australia; Dinaciclib treatment also rapidly suppressed Mcl-1 protein 4Cancer Epigenetics Laboratory, Research Division, Peter MacCallum expression, with no discernible reduction in Bcl-2 or Bcl-xL protein Cancer Centre, Melbourne, Victoria, Australia and observed in murine (Figure 1e) or human (Figure 1f) cells. To 5School of Clinical Sciences at Monash Health, Monash University, determine the functional importance of Mcl-1 in regulating Clayton, Victoria, Australia dinaciclib-mediated apoptosis, a representative Eμ-Myc lymphoma E-mail: [email protected] was stably transduced to express Mcl-1 off a retroviral promoter. 6These authors contributed equally to this work. As shown in Figure 1g, exogenously expressed Mcl-1 significantly protected Eμ-Myc cells from dinaciclib-induced apoptosis. The in vivo efficacy of dinaciclib was then assessed by REFERENCES transplanting the same Eμ-Myc lymphomas into cohorts of 1 Savage KJ, Johnson NA, Ben-Neriah S, Connors JM, Sehn LH, Farinha P et al. MYC syngeneic C57Bl/6 recipients. Compared with the vehicle control, gene rearrangements are associated with a poor prognosis in diffuse large B-cell dinaciclib treatment was well tolerated and associated with a lymphoma patients treated with R-CHOP chemotherapy. Blood 2009; 114: highly significant survival advantage of tumor-bearing mice, 3533–3537. including those bearing a p53-null lymphoma and a lymphoma 2 Hu S, Xu-Monette ZY, Tzankov A, Green T, Wu L, Balasubramanyam A et al. MYC/BCL2 protein coexpression contributes to the inferior survival of activated with a spontaneous p53 mutation encoding a dominant-negative B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene p53 protein (Figures 2a–c, Supplementary Figure S3). In contrast, expression signatures: a report from The International DLBCL Rituximab-CHOP dinaciclib-mediated therapeutic efficacy was severely attenuated Consortium Program. Blood 2013; 121: 4021–4031. in isogeneic p53-competent Eμ-Myc lymphoma overexpressing 3 Kanazawa S, Soucek L, Evan G, Okamoto T, Peterlin BM. c-MYC recruits P-TEFb for Mcl-1 (Figure 2d). In separate experiments, mice bearing transcription, cellular proliferation and apoptosis. Oncogene 2003; 22: 5707–5711. transplanted Eμ-Myc cells were left untreated for 12 days to 4 Marshall NF, Peng J, Xie Z, Price DH. Control of RNA polymerase II elongation establish bulky nodal disease, at which time they received a single potential by a novel carboxyl-terminal domain kinase. J Biol Chem 1996; 271: dose of dinaciclib or vehicle 1 or 4 h before being killed and 27176–27183. before the lymph nodes were harvested. Consistent with the 5 Gargano B, Amente S, Majello B, Lania L. P-TEFb is a crucial co-factor for Myc transactivation. Cell Cycle 2007; 6: 2031–2037. in vitro data, lymph node protein lysates showed reductions of 6 Cowling VH, Cole MD. The Myc transactivation domain promotes global phos- pRpb1 and total Mcl-1 protein (Figure 2e), concomitant with the phorylation of the RNA polymerase II carboxy-terminal domain independently of induction of apoptosis (Supplementary Figure S4). Finally, direct DNA binding. Mol Cell Biol 2007; 27: 2059–2073. dinaciclib treatment of immunocompromised mice xenografted 7 Huang CH, Lujambio A, Zuber J, Tschaharganeh DF, Doran MG, Evans MJ et al. with the human IG-cMYC-translocated lymphoma was associated CDK9-mediated transcription elongation is required for MYC addiction in with reduced disease progression and significantly prolonged hepatocellular carcinoma. Genes Dev 2014; 28: 1800–1814. overall survival (Figures 2f and g). 8 Parry D, Guzi T, Shanahan F, Davis N, Prabhavalkar D, Wiswell D et al. Dinaciclib In conclusion, our findings indicate that CDK9 inhibition by (SCH 727965), a novel and potent cyclin-dependent kinase inhibitor. Mol Cancer – dinaciclib is highly effective in aggressive MYC-driven lymphomas, Ther 2010; 9: 2344 2353. fi 9 Adams JM, Harris AW, Pinkert CA, Corcoran LM, Alexander WS, Cory S et al. The including ‘poor-risk’ p53-de cient clones, via selective inhibition of c-myc oncogene driven by immunoglobulin enhancers induces lymphoid critical MYC targets including Mcl-1 (which is currently undrug- malignancy in transgenic mice. Nature 1985; 318: 533–538. 13,14 gable with existing BH3 mimetics). Our data suggest a linear 10 Mason KD, Vandenberg CJ, Scott CL, Wei AH, Cory S, Huang DCS et al. In vivo and druggable dependency between MYC and Mcl-1 that is efficacy of the Bcl-2 antagonist ABT-737 against aggressive Myc-driven lympho- contingent on CDK9 signaling. These findings are of particular mas. Proc Natl Acad Sci USA 2008; 105: 17961–17966. interest in the context of a recent publication by Kelly et al.,15 11 Raje N, Kumar S, Hideshima T, Roccaro A, Ishitsuka K, Yasui H et al. Seliciclib further highlighting the dependency of MYC-driven B-cell (CYC202 or R-roscovitine), a small-molecule cyclin-dependent kinase inhibitor, lymphoma to Mcl-1. Rapid clinical translation of CDK9 inhibitors mediates activity via down-regulation of Mcl-1 in multiple myeloma. Blood 2005; 106: 1042–1047. to MYC-dysregulated lymphoid malignancy should now be 12 Lacrima K, Valentini A, Lambertini C, Taborelli M, Rinaldi A, Zucca E et al. In vitro considered. activity of cyclin-dependent kinase inhibitor CYC202 (Seliciclib, R-roscovitine) in mantle cell lymphomas. Ann Oncol 2005; 16: 1169–1176. 13 Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA et al. CONFLICT OF INTEREST An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature The authors declare no conflict of interest. 2005; 435: 677–681.

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Letters to the Editor 1441 14 Souers AJ, Leverson JD, Boghaert ER, Ackler SL, Catron ND, Chen Jet al. ABT-199, This work is licensed under a Creative Commons Attribution- a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing NonCommercial-NoDerivs 4.0 International License. The images or platelets. Nat Med 2013; 19: 202–208. other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under 15 Kelly GL, Grabow S, Glaser SP, Fitzsimmons L, Aubrey BJ, Okamoto T et al. the Creative Commons license, users will need to obtain permission from the license Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when holder to reproduce the material. To view a copy of this license, visit http:// they bear mutations in p53. Genes Dev2014; 28: 58–70. creativecommons.org/licenses/by-nc-nd/4.0/

Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu)

Targeting PD1 –PDL1 immune checkpoint in plasmacytoid dendritic cell interactions with T cells, natural killer cells and multiple myeloma cells

Leukemia(2015) 29, 1441–1444; doi:10.1038/leu.2015.11 PDL1, whereas T cells showed high PD1 levels (Figures 1a–c). No significant PDL1 expression was noted on normal BM plasma cells. Our findings are consistent with previous reports showing that 3,10–13 Despite the advent of bortezomib, thalidomide and lenalidomide, MM cells, but not normal plasma cells, express PDL1. These relapse of multiple myeloma (MM) is common, and novel data indicate that the interactions between PDL1-expressing MM therapies are needed urgently.1 Interactions of MM cells with cells and pDCs with PD1-positive T cells may contribute to both bone marrow (BM) accessory and immune e ector cells inhibit T-cell and pDC immune dysfunction in MM, and MM cells may antitumor immunity as well as induce MM growth, survival and escape antitumor immunity by virtue of PDL1 expression. drug resistance.1 For example, we showed that plasmacytoid We next examined whether blockade of PDL1–PD1 restores dendritic cells (pDCs) are increased in the BM of MM patients anti-MM immune response and/or a ects pDC-induced MM cell compared with normal BM, and these contribute to immune growth, using a monoclonal antibody (Ab) specifically directed dysfunction, as well as promote tumor cell growth and survival.2 against PDL1. A recent study analyzed the expression of PD1 and Aberrant pDCs’ function in MM is evidenced by their interaction PD1-ligands in the tumor immune microenvironment and not only with MM cells but also with immune e ectorTcells: MM demonstrated clinical responses to anti-PD1 Ab therapy in PDL1- positive tumors.8 PDL1 is expressed in both pDCs and MM cells, BM pDCs have decreased ability to trigger T-cell proliferation 13 compared with normal pDCs.2 Dysfunctional T cells and natural including relapsed or refractory MM, and we hypothesize that killer (NK) cells in MM3,4 together with functionally defective blockade of PDL1 will alleviate T-cell immune suppression 2 conferred by both MM cells and pDCs during pDC–MM–T cell pDCs confer immune suppression in MM. To date, the mechan- interactions. Moreover, as PDL1 binds not only to PD1 but also to ism(s) and the role o mmunoregulatory molecules mediating CD80, on T cells to induce T-cell inhibition,14 anti-PDL1 Ab may pDC–T cell and pDC–NK cell interactions in MM remain undefined. block both co-inhibitory signals on T cells. Preclinical and clinical Here we extended our previous studies2,5 to examine the role of studies have begun to examine the utility of anti-PDL1 immune checkpoint receptor programmed cell death protein 1 monoclonal Ab in MM.10,11,15 Here we targeted PDL1 rather than (PD1) and its ligand PDL1 in pDC–T cell and pDC–NK cell PDL2 for the following reasons: (1) PDL1 is more restricted in its interactions in the MM BM milieu, and to determine whether this expression on normal tissues than PDL2, and targeting PDL1 may interaction represents a therapeutic target to restore antitumor therefore cause less on-target o -tissue toxicity;9 (2) a recent immunity and cytotoxicity. report correlated PDL1, but not PDL2, expression with response to PD1 (CD279), a member of the CD28 family of receptors, is anti-PD1 therapy;8 and (3) we found that both pDCs and MM cells expressed on the surface of antigen-activated and -exhausted T 4 express variable and low levels of PDL2 versus PDL1. cells. PD1 has two ligands, PDL1 (B7-H1; CD274) and PDL2 (B7-DC; We first examined whether blockade of PDL1 a ects the ability CD273). Although PDL1 expression has not been observed in of pDC to induce MM cell growth. The patient MM cells or MM cell normal epithelial cells, it is highly expressed on many solid 6 lines (MM.1S, MM.1R and RPMI-8226) were cultured either alone or tumors. PDL2 is more broadly expressed on normal healthy together with MM–pDCs in the presence or absence of anti-PDL1 tissues than PDL1. The physiological role of PD1 is to maintain Ab for 72 h, followed by analysis of growth. pDCs triggered T-cell homeostasis by restricting T-cell activation and proliferation, proliferation of autologous MM cells and MM cell lines, as in our thereby preventing autoimmunity. Importantly, the interaction previous studies.2,5 Importantly, anti-PDL1 Ab did not significantly of PD1+ T cells with PDL1-expressing cells inhibits T-cell inhibit pDC-triggered growth of MM cells (Figure 1d and 7–9 responses. In the context of MM, studies have demonstrated Supplementary Figure 1). Our recent study showed that targeting PD1-expressing T cells and NK cells in the MM BM milieu, as well toll-like receptor-9 blocks pDC-induced MM cell growth,2,5 which as PDL1 on MM cells.3,10–13 However, the expression of PDL1–PD1 served as a positive control in these studies (Figure 1d and on MM patient-derived pDCs and its functional significance during Supplementary Figure 1). Although blocking PDL1 does not a ect pDC–MM–T–NK cell interactions remain undefined. pDC-induced MM cell growth, pDC–MM cell interactions upregu- We first analyzed freshly isolated MM cells, pDCs and T cells late PDL1 expression on both cell types, consistent with earlier from MM patient BM samples (n = 11) for PDL1 and PD1 observations that BM stromal cells induce PDL1 expression on MM expression using flow cytometry (fluorescence-activated cell sorter cells.13 Such interactive mechanisms enhancing PDL1 expression (FACS)). Both MM cells and pDCs expressed high surface levels of in the MM BM milieu further abrogate PD1-expressing T-cell

Accepted article preview online 24 February 2015; advance online publication, 24 February 2015

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SUPPLEMENTARY INFORMATION (Gregory et al. 2015)

METHODS Cell lines Eµ-Myc lymphomas and Burkitt cell lines were derived and cultured as previously described.1 Retroviral transduction of Eµ-Myc lymphoma and BL-41 cells with the murine stem cell virus-internal ribosomal entry site-green fluorescence protein (MSCV- I-GFP) Mcl-1 and Luc2 constructs and the TRMPVIR construct were performed as previously described.1

In vitro cell death assays Dinaciclib was provided by Merck Research Laboratories (Boston, USA) and reconstituted with dimethylsulfoxide (DMSO) into 10mM stocks for in vitro use. Lymphoma cells were incubated then analyzed for annexin-V and propidium iodide staining as previously described.1

Western blots Cell lysate preparation, SDS polyacrylamide gel electrophoresis and immunoblotting was performed according to standard techniques1 with the following primary antibodies: Mcl-1 (Rockland Immunochemicals Inc., Gilbertsville, PA), Bcl-2 (BD Pharmingen, San Jose, CA), HSP90 (AC88, Enzo Life Sciences, Inc., Farmingdale, NY), β-actin (Sigma-Aldrich, St. Louis, MO), α-tubulin (Merck KGaA, Darmstadt, Germany),

Bcl-2 and Bcl-xL (Santa Cruz Biotechnology, Inc., Dallas, TX), Mcl-1, c-Myc, pRpb1 CTDSer2/5, pRpb1 CTDSer2 and pRpb1 CTDSer5 (Cell Signaling Technology, Danvers, MA).

RNA isolation and quantitative real-time PCR

RNA was isolated from treated cells or vehicle controls using the NucleoSpin® RNA extraction kit, (Macherey-Nagel, Bethlehem, PA) and qPCR was performed as previously described using SYBR green stain (Invitrogen, Life Technologies, Victoria, Australia), with GAPDH and L32 as the murine and

108 Chapter 3 human control genes, respectively.2 Primer sequences were: Mus musculus Mcl- 1 F: GGTGCCTTTGTGGCCAAACACTTA R: ACCCATCCCAGCCTCTTTGTTTGA, Mus musculus Bcl-2 F: ATGACTGAGTACCTGAACCGGCAT R: GGGCCATATAGT TCCACAAAGGCA, Mus musculus GAPDH F: CCTTCATTGACCTCAACTAC R: GGAAGGCCATGCCAGTGAGC, Homo sapiens Mcl-1 F: AACAAAGAGGCTGGGATGGGTTTG R: AAACCAGCTCCTACTCCAGCAACA, Homo sapiens c-MYC F: GGACGACGAGACCTTCATCAA R: CCAGCTTCTCTGAGACGAGCTT, Homo sapiens L32 F: TTCCTGGTCCACAATGTCAAG R: TTGTGAGCGATCTCGGCAC.

Chromatin immunoprecipitation assay and real-time PCR Eµ-Myc lymphomas cells were cultured as described above. Chromatin was prepared and immunoprecipitated as previously described3, except for the following differences: Cells were sonicated using a Covaris S2 (Covaris Inc., Woburn, MA), antibodies used were pRpb1 CTDSer2 (Cell Signaling Technology, Danvers, MA) and normal rabbit IgG isotype control (Santa Cruz Biotechnology, Inc., Dallas, TX), bound using poly-A beads (nProtein A Sepharose 4 Fast Flow, GE Healthcare, Feiburg, Germany) and DNA purification was performed using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel, Bethlehem, PA) prior to RT-PCR being performed as described above.

The following primer sets were used: Mcl-1 set 1 F: TTCCTCACTCCTGACTTCCG R: CCAAACATGGTCGGACGC, Mcl-1 set 2 F: TGTAAGGACGAAACGGGACT R: CACCCCATTTCCACTCCACG, Mcl-1 set 3 F: TAGAGATGGAAGAGGGGCCAG R: TAGGGCTTCTCTCTCAACACTC.

In vivo assays All animal studies were performed in accordance with national and institutional ethical requirements. Dinaciclib was reconstituted in 20% hydroxypropyl beta cyclodextrin (HPBCD) then sonicated into solution. Syngeneic C57Bl/6 mice were injected with 1x105 Eµ-Myc lymphoma cells and NOD-scid IL2Rγnull mice were injected with 1x106 BL-41 cells 3 days prior to commencing therapy with dinaciclib (30mg/kg) or 20% HPBCD, twice weekly by intraperitoneal injection. Full blood count analysis was performed

109 Chapter 3 on the CELL-DYN Sapphire Blood Analysis Instrument (Abbott Laboratories, Abbott Park, IL). In vivo imaging was performed using the Xenogen IVIS 100 Imaging System (PerkinElmer Inc., Boston, MA). TUNEL assay was performed on formalin-fixed paraffin embedded tissue using the ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Merck KGaA, Darmstadt, Germany) and images were acquired using the Olympus BX-51 microscope (Olympus, Tokyo, Japan) with SPOT software (Sterling Heights, MI).

Statistical analysis Statistical analysis was performed using GraphPad Prism Software, Version 6.0c (La Jolla, CA).

SUPPLEMENTARY REFERENCES 1. Shortt J, Martin BP, Newbold A, et al. Combined inhibition of PI3K-related DNA damage response kinases and mTORC1 induces apoptosis in MYC- driven B-cell lymphomas. Blood. 2013;121(15):2964–2974. 2. Waibel M, Solomon VS, Knight DA, et al. Combined targeting of JAK2 and Bcl-2/Bcl-xL to cure mutant JAK2-driven malignancies and overcome acquired resistance to JAK2 inhibitors. Cell Rep. 2013;5(4):1047–1059. 3. Dawson MA, Bannister AJ, Göttgens B, et al. JAK2 phosphorylates histone H3Y41 and excludes HP1α from chromatin. Nature. 2009; 461(8):819-822.

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a b c #6066.TRMPVIR.shp53 #4242 #3391 #6066.TRMPVIR.shp53 Dox off Dox on p53 WT p53-/- Dox off Dox on

* * NIH/3T3 #4242

** 100 100 sh p53 sh Renilla sh Scrambled sh p53 sh Renilla sh Scrambled

50 50 p53 53 kDa % Annexin / PI % Annexin / PI 0 Actin 45 kDa 0 0 4 8 16 0 4 8 16 0 0 0 4 8 16 16 Dinaciclib [nM] Etoposide Etoposide Etoposide Etoposide

Dinaciclib [nM] Supplementary Figure S1. (a) Dinaciclib potently induces apoptosis of E -Myc lymphoma #6066 with doxycycline-inducible shp53 irrespective of p53 activity. E - Myc lymphomas #6066 (with or without doxycycline, Dox), #4242 (wild-type p53) and #3391 (p53 null) were cultured in vitro with DMSO vehicle control, etoposide 20nM positive control (red bars) or dinaciclib for 24 h then analyzed by flow cytometric analysis for annexin-V / propidium iodide (PI) positivity. *p<0.05 for etoposide controls. (b) Western blot showing inducible reduction of p53 expression in E -Myc lymphoma #6066 used in Supplementary Figure S1a. -Actin loading control shown. (c) NIH/3T3 murine fibroblast cells were cultured in vitro with DMSO vehicle control or dinaciclib for 24 h then analyzed by flow cytometric analysis for annexin-V / propidium iodide (PI) positivity. **p<0.001 comparing treatments at 16nM concentration.

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c-MYC Mcl-1

BL-41 Ramos BL-41 Ramos * * * * 1.0

0.5

0 Relative mRNA level (DMSO)

DMSO DMSO DMSO DMSO

Dinaciclib Dinaciclib Dinaciclib Dinaciclib Supplementary Figure S2. Mcl-1 and c-MYC mRNA expression in human IG- cMYC-translocated BL-41 and Ramos cell lines following 2 h in vitro treatment with DMSO or 20nM dinaciclib. Transcript levels are represented as fold change compared with DMSO. *p<0.01.

a b c NS 200 6 * 1000 ** /L) 9 /L) 9

100 3 500 ophils (x10 Hemoglobin (g/L) Platelets (x10 0 Neut r 0 0

ehicle ehicle ehicle V V V

Dinaciclib Dinaciclib Dinaciclib Supplementary Figure S3. Dinaciclib therapy is associated with minimal hematological toxicity in vivo. Mice from experiments shown in Figure 2a-d were bled at day 12 post-transplantation. Hemoglobin concentration (a), neutrophil (b) and platelet counts (c) shown. NS not significant, *p<0.01, **p<0.0001.

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a c 50 Vehicle Dinaciclib

25 % PI positive H & E 0

ehicle V

Dinaciclib 1 h Dinaciclib 4 h b

100 TUNEL G2-M

S 50 G1

Sub-G1

% Cells in cell cycle 0

ehicle V

Dinaciclib 1 h Dinaciclib 4 h

Supplementary Figure S4. Dinaciclib therapy rapidly induces apoptosis of E -Myc lymphoma in vivo. Cell suspensions were prepared from lymph nodes of C57Bl/6 mice 12 days following transplantation with E -Myc lymphoma #4242 and 1 or 4 hours following a single dose of dinaciclib (30mg/kg) or 20% HPBCD. Flow cytometric analysis was performed for (a) loss of viability by propidium iodide (PI) uptake and (b) nuclear DNA content by Nicoletti stain. (c) Representative lymph node sections from mice at 4 h timepoint described in Supplementary Figure S3a-b showing induction of apoptosis with dinaciclib treatment according to morphology (pyknotic nuclei in H & E section) and by TUNEL assay.

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3.3 Discussion

3.3.1 Dinaciclib as a potent inhibitor of Pol II activation

As hypothesised, the CDK9-inhibitor dinaciclib mediated potent dephosphorylation of Serine 2 within heptapeptide repeats of the Pol II CTD with the levels of Serine 5 phosphorylation relatively unaffected (Gregory et al. 2015). This observed suppression of Pol II activation was also seen to transcriptionally reduce Mcl- 1 mRNA preferentially upon short incubations and is consistent with historical and seliciclib (R-Roscovitine) (Gojo et al. 2002; MacCallum et al. 2005; Lacrima et biomarkers of CDK9 inhibition mediated by pan-CDK inhibitors such as flavopiridol al. 2005; Gao et al. 2006; Cirstea et al. 2013).

3.3.2 Dinaciclib potently induces apoptosis of MYC-driven B-cell lymphoma

Global transcriptional repression through inhibition of CDK9-mediated Pol II transcriptional elongation can elicit a multitude of signalling aberrations. However, the very short half-life of Mcl-1 (Cuconati et al. 2003; Nijhawan et al. 2003) is critical to the apoptotic potential of CDK9 inhibition. In hindsight it is hardly surprising that such a potent inhibitor of CDK9 would be able to rapidly induce apoptosis in an Mcl-1-dependent manner. Our hypothesis of a druggable linear oncogenic signalling pathway (Figure 3.9) requiring CDK9-mediated activation within this chapter. of Pol II to effect Mcl-1 transcription has been supported by experimental findings

Recent genetic studies highlighting the significant oncogenic dependency of MYC- observed in our models (Kelly et al. 2014; Aubrey et al. 2015). By interfering driven lymphoma on Mcl-1 further provide rationale for the therapeutic efficacy directly with anti-apoptotic protein expression to induce programmed cell death, it is also not surprising that dinaciclib is able to effect apoptosis in a p53- independent manner. This is of critical importance for clinical development, as it has been shown that p53 mutation confers poor prognosis to aggressive MYC- driven lymphoma, and typically renders resistance to standard chemotherapy and radiotherapy (Ichikawa et al. 1997; Xu-Monette et al. 2012; Leroy et al. 2002; Kerbauy et al. 2015).

The cytostasis observed in apoptosis-protected cells also provided insight into other biological effects of dinaciclib. While these observations may correspond with inhibition of ‘cell cycle’-related CDKs such as CDK1 and CDK2, another possibility is that CDK9 inhibition by dinaciclib may interfere with transcription

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Dinaciclib

PTEFb Cyclin T1 CDK9 MYC YSPTSPS/YSPTSPS RNA polymerase II

Transcription

Mcl-1

Figure 3.9: CDK9-inhibition with dinaciclib reduces Pol II-mediated transcription of critical MYC targets including MCL-1

Schematic representation of the proposed apoptotic mechanism of dinaciclib Myc lymphoma. CDK9 inhibition with dinaciclib leads to reduced phosphorylation of Pol II (Serine 2) with an associated transcriptional in the treatment of Eμ- reduction of anti-apoptotic MCL-1.

115 Chapter 3 of the activating cyclin binding partners for these cell cycle kinases. Furthermore, p53 activation may also be contributing to the G2/M-arrest in this p53-competent clone. This was not pursued further as the spontaneously-arising lymphomas had already been shown to be undergoing apoptosis in the previous assays, indicating programmed cell death that was already occurring. Repeating these experiments that this cytostatic effect was of lesser significance when compared to the with overexpression of anti-apoptotic Bcl-2 in a p53-mutated / null clone would have been of value in order to delineate whether p53 is playing any role in the observed cytostasis.

(Parry et al. 2010), it should be noted that kinome inhibitory assays have also Despite an emphasis on the CDK-inhibitory profile of dinaciclib in the literature revealed other non-CDK targets of dinaciclib including bromodomain proteins (Martin et al. 2013) and GSK3 activity (Wang and Gray 2013), among others. studies of GSK3 inhibitors for the treatment of AML. GSK3 inhibition in mixed These findings are of particular interest given the recent success of preclinical lineage leukaemia (MLL)-rearranged AML has been shown to reduce GSK3- mediated destabilisation of p27Kip1, thereby restoring inhibition of cyclin D-CDK4 and cyclin E-CDK2 complex-mediated cell cycle progression (Wang et al. 2008). These conclusions also propose dinaciclib-mediated GSK3 inhibition as a possible contribution to the cytostatic effect observed in vitro.

However, GSK3 has also been shown to play a critical role in direct regulation of Mcl-1 expression across a variety of non-haematological and haematological cell lines, including murine pro-B precursor cells. GSK3 phosphorylates Mcl-1 (Ser 159), leading to increased ubiquitinylation and degradation of Mcl-1 (Maurer et al. 2006). This process was demonstrated using models of precursor B-cells and could be rescued by pharmacological inhibition of GSK3 or generation of a phosphorylation site mutation (S159A). Furthermore, arsenic trioxide treatment of acute promyelocytic leukaemia cells has been shown to remove AKT-mediated repression of GSK3, leading to increased pMcl-1 (Ser 159) with resultant apoptosis activity by dinaciclib would stabilise Mcl-1 through reduction in pMcl-1 (Ser induction (Wang et al. 2013b). These findings suggest that inhibition of GSK3 159), conferring protection from ubiquitinylation and proteasomal degradation whether dinaciclib-mediated inhibition of GSK3 is leading to reduction in pMcl- and leading to an anti-apoptotic effect. It would have been difficult to assess 1 (Ser 159) in Eµ-Myc lymphoma due to the potent transcriptional suppression of Mcl-1 expression. However, assessing the effect of dinaciclib on pMcl-1 (Ser 159) in Eµ-Myc cells transduced for forced overexpression of Mcl-1 would have

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regulation of Mcl-1. been beneficial in order to investigate the effect dinaciclib is having upon GSK3 Finally, despite a principal association of CDK1 with cell cycle progression, CDK1 inhibition in the context of MYC-dysregulated malignancy has previously been shown to downregulate survivin to precipitate apoptosis (Goga et al. 2007). It would be useful to repeat the Western blot experiments in order to assess whether dinaciclib is effecting any change of survivin expression. While it is possible and targets in addition to CDK9, the studies showing protection to Eµ-Myc lymphoma even probable that dinaciclib is mediating efficacy through inhibition of other with forced overexpression of Mcl-1 certainly implicate Mcl-1 as a critical determinant of dinaciclib-mediated apoptosis.

3.3.3 Dinaciclib is a tolerable and effective therapy against MYC-driven B-cell lymphoma in vivo

The observed in vivo curative responses in subsets of tumour-bearing mice, far exceeded expectation efficacy of dinaciclib, and particularly the ability to induce than any other targeted therapy observed in the Eµ-Myc model to date in our for a targeted therapeutic in a chemotherapy-free protocol. This efficacy is greater laboratory (with comparative examples of ibrutinib and fedratinib shown). Of to treated mice in every therapy experiment when compared to vehicle-treated particular interest, dinaciclib was able to confer a significant survival advantage controls. No assessed clones were found to be completely refractory to up-front dinaciclib therapy.

3.3.4 Dinaciclib effects on MYC expression

The main discordance observed with regard to experiments in this chapter were those of dinaciclib on MYC expression between the murine and human cell lines. Whilst dinaciclib potently suppressed MYC mRNA levels and MYC protein expression in the human IG-cMYC-translocated Burkitt lymphoma cell lines at short timepoints (Gregory et al. 2015), no such change was observed upon interrogation of Eµ-Myc lymphoma. This is likely explained by the nature of the latter model. Having been derived from transgene insertion in founder mice, it is likely that transgenic Myc expression is subject to different epigenetic and transcriptional regulation than endogenous Myc. This is consistent with work performed in our laboratory with the bromodomain inhibitor, JQ1, whereby short incubations with Eµ-Myc lymphoma lead to rapid and potent suppression of endogenous Myc mRNA, with no discernible effect on transgenic Myc mRNA expression (Hogg et al. 2016).

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Whether the potent suppression of MYC observed in the dinaciclib studies with Burkitt lymphoma are mediated by CDK9 inhibition or by other ‘off-target’ inhibition or indirect effects is of particular interest, given that it has also been recently shown that higher concentrations of dinaciclib can directly mediate bromodomain inhibition (Martin et al. 2013). If time and resources permitted, repeating the qRT-PCR analysis of dinaciclib-treated Eµ-Myc cells to assess for relative effects on each of endogenous and transgenic Myc mRNA levels would be useful to determine if endogenous Myc is indeed repressed, and the maintained protein expression observed resulting entirely from unaffected transgenic Myc expression. Furthermore, a global analysis using RNA-sequencing would enable a hypothesis-free assessment of other dinaciclib-mediated transcriptional targets that may be contributing to the potent apoptosis induction. Further interrogation of experiments in the following results chapter. as to which dinaciclib targets are critical for observed efficacy is a major component 3.3.5 Conclusion

It can be concluded that dinaciclib therapy represents an effective novel therapeutic strategy against MYC-driven B-cell lymphoma. The timeliness of studies demonstrating the Mcl-1 dependency of MYC-driven B-cell lymphoma (Kelly et al. 2014; Aubrey et al. 2015), and the fact that direct therapeutic targeting of Mcl-1 has been elusive, further enhances the potential value that this targeted therapy could provide in the clinic.

118 Chapter 4: Genetic and pharmacologic targeting of CDK9 in MYC-driven models of B-cell lymphoma

119 Chapter 4

4.1 Introduction

dinaciclib in the treatment of MYC-driven B-cell lymphoma both in vitro and in Experimental work presented in chapter three demonstrated the efficacy of vivo, with associated reductions in Pol II activation and Mcl-1 expression. These

2) phosphorylation. However, published kinome inhibitory data of dinaciclib findings are hypothesised to result from inhibition of CDK9-mediated Pol II (Ser and many other CDK and non-CDK molecules including BET proteins targeted at confirms lack of specificity, with nanomolar potency against CDK1, 2, 5 and 9, micromolar concentrations (Parry et al. 2010; Wang and Gray 2013; Martin et al. 2013).

Whether the biologically potent responses to dinaciclib are the result of CDK9 inhibition alone or the synergistic antagonism of other targets in combination with CDK9 remains an unanswered question. In order to interrogate this, experimental work in chapter four utilises further pharmacologic inhibitors of CDK9 and other CDKs to assess whether their ability to induce apoptosis and associated signalling changes is consistent with previous observations using dinaciclib. We show that novel and selective CDK9 inhibitors in preclinical development are shown to elicit similar effects upon Pol II activation and Mcl-1 expression, though inconsistent effects on Myc expression and cytostasis.

A series of genetic experiments are described in this chapter to assess the effect of identify the CDKs that are critical for MYC-driven B-lymphoma maintenance. CDK9 specific CDK9 depletion. In addition a boutique gene knockdown screen is used to depletion is demonstrated to phenocopy pharmacologic CDK9 inhibition and supports the hypothesis of a linear druggable pathway (Figure 4.1). Furthermore, model and for which relatively little is published regarding function and regulation. the boutique screen identifies novel CDKs that are functional dependencies of this

Finally, experimental work is presented that is aimed to assess whether CDK9 inhibitor-mediated repression of Mcl-1 would synergise with Bcl-2 antagonism for potent apoptosis induction. In vivo studies demonstrate a surprising and previously unreported acceleration of MYC-driven disease from Bcl-2 antagonism alone, which may have implications for this class of therapeutic as its use is expanded in lymphoma (Roberts et al. 2016). the clinic following the efficacy shown in clinical trials for the treatment of indolent

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CDK9 inhibitors

PTEFb Cyclin T1 CDK9 MYC YSPTSPS/YSPTSPSCTD RNA polymerase II

Transcription

Mcl-1

Figure 4.1: CDK9 regulates full activation of Pol II-mediated transcription of critical MYC targets including MCL-1

Schematic representation of the proposed oncogenic pathway demonstrating MYC recruitment of cyclin T1 to recruit P-TEFb to MYC transcriptional target sites. CDK9 inhibitors (red line) or genetic interference of CDK9 (red cross) are predicted to phenocopy the effects of dinaciclib, including reduced phosphorylation of RNA Polymerase II, subunit B1 at serine residues at position two within heptapeptide repeats in the carboxy-terminal domain (CTD), with an associated reduction in MCL-1. P denotes phosphorylation.

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4.2 Results

4.2.1 CDK9 inhibitors potently induce apoptosis of Eµ-Myc lymphoma and human IG-cMYC-translocated lymphoma in vitro

In order to assess whether induction of apoptosis with dinaciclib was mediated through inhibition of CDK9 or through other CDK or non-CDK targets, additional Myc and human IG-cMYC-translocated lymphoid cell lines in vitro. The kinome inhibitory CDK inhibitors with more selective target profiles were tested using the Eµ- Table 1.1. profiles of these inhibitors are listed in Incubation of Eµ-Myc

lymphoma cell lines with the prototypical flavonoid pan-CDK at nanomolar concentrations approaching those quoted for the biochemical IC inhibitor, flavopiridol, induced apoptosis in a p53- and Bim-independent manner50 CDK9 (Table 1.1, Figure 4.2a-c). Similar to dinaciclib, higher concentrations of induced overexpression of anti-apoptotic Mcl-1 or Bcl-2 (Figure 4.2d,e). flavopiridol were required for apoptosis induction in the setting of retrovirus-

A similar suite of Eµ-Myc lymphoma cell lines were next exposed to a novel CDK9 inhibitor, A1592668.1 (AbbVie, Boston, MA) and induction of apoptosis was assessed. As seen in Table 1.1, this inhibitor shows a more narrow selectivity

50 less than for cyclin-dependent kinases than dinaciclib or flavopiridol with an IC induced apoptosis in Eµ-Myc cell lines in a p53-independent manner at nanomolar 100nM for CDK9 and CDK4. Similar to dinaciclib and flavopiridol, this inhibitor concentrations consistent with CDK9 inhibition (Figure 4.3a,b). Furthermore, apoptosis induction was observed to be independent of functional Bim or Bmf (Figure 4.3c,d). Forced overexpression of Mcl-1 and Bcl-2 again conferred relative protection against A1592668.1-mediated apoptosis (Figure 4.3e,f).

In contrast to the transcriptional activity of CDK9, CDK4 is a cell-cycle related kinase and is involved in cell cycle G1- transition. The CDK4 / cyclin D heterodimer is involved in phosphorylation of the retinoblastoma protein (Rb), leading to dissociation of pRb from E2 promoter-binding-protein-dimerization partners (E2F-DP) (Kato et al. 1993; Ewen et al. 1993). This removes suppression of E2F-DP transcriptional activity with resultant transition into S-phase. Therefore, it would be unlikely that the CDK4 inhibitory properties of A1592668.1 would be responsible for the apoptosis that is observed with Eµ-Myc lymphoma. In order to further show that CDK4 is not likely to be the target mediating A1592668.1- induced apoptosis, a further selective CDK4/6-inhibitor, palbociclib (Pfizer, La 122 Chapter 4

Jolla, CA), was used.

As can be seen from Table 1.1, palbociclib has an IC50 of 11nM and 16nM for CDK4 and CDK6, respectively. Palbociclib has not been shown to target transcriptional

CDKs and while the IC50 for CDK9 has not been published, CDK9 activity has been shown to be reduced to 27% of untreated control in a cell free inhibitory assay following one hour incubation at 10µM concentration (Liu and Gray 2015). When Eµ-Myc and palbociclib in apoptosis assays, potent induction of apoptosis was again seen lymphoma cells were exposed to the CDK inhibitors dinaciclib, flavopiridol

(Figure 4.4a). Of note, however, nanomolar concentrations of palbociclib were with dinaciclib and flavopiridol, but not with palbociclib at assessed concentrations shown to induce cytostasis with a significant increase in proportion of cells in G1- analysis (Figure 4.4b,c). This cytostasis without apoptosis is consistent with phase and significant reduction in S-phase of the cell cycle according to cell cycle CDK4/6 inhibition of palbociclib, and essentially excludes CDK4-inhibition as the apoptotic mechanism of A1592668.1. Therefore, CDK9 is further implicated as the critical target of A1592668.1-mediated apoptosis using the Eµ-Myc system.

A second novel CDK9 inhibitor, AZ-CDK9 [Table 1.1, also known as AZ’5576 (Cidado et al. 2016) and PC585 (Garcia-Cuellar et al. 2014)], was sourced from Astra- Zeneca (Boston, MA) and assessed for induction of apoptosis of Eµ-Myc lymphoma to further pharmacologically validate CDK9 as a critical target. As seen in Figure 4.5a, and consistent with the other assessed inhibitors, AZ-CDK9 induced apoptosis at low nanomolar concentrations in a p53-independent manner, with abrogation of apoptosis seen with lymphoma cell lines overexpressing murine Mcl-1 and Bcl- 2. In order to further assess the effect of anti-apoptotic protein overexpression, a number of Eµ-Myc lymphoma cell lines were derived to overexpress select members of the homo sapiens anti-apoptotic BCL-2 family driven by retroviral promoter expression; BCL-2, MCL-1, BCL-XL, A1 and BCL-W. These cell lines were derived using homo sapiens rather than mus musculus members in order to create more relevant models for direct BCL-2 family antagonism described in chapter Myc lymphoma #4242, forced overexpression of any anti-apoptotic family member (BCL-2, MCL- five. Upon exposure to AZ-CDK9, and in contrast to the parental Eµ- 1, BCL-XL, A1 or BCL-W) was associated with relative protection against CDK9- mediated apoptosis induction (Figure 4.5b,c).

Cell cycle analysis was next performed with the two selective CDK9-inhibitors, A1592668.1 (Figure 4.6a) and AZ-CDK9 (Figure 4.6b), using apoptosis-protected Eµ-Myc lymphoma overexpressing Bcl-2. Similar to dinaciclib, both inhibitors

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Figure 4.2: Flavopiridol induces apoptosis of Eµ-Myc lymphoma in vitro

(a Myc lymphoma cell line #4242 was cultured for 24 hours in vitro ) Exponentially growing Eμ- vehicle as represented by 0nM. Flow cytometric analysis was performed at 24 with varying concentrations of flavopiridol (20 – 156nM) or DMSO hours to assess for annexin-V / PI uptake. (b) Exponentially growing p53-null Myc lymphoma cell line #3391 was cultured for 24 hours in vitro with varying

Eμ- 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V concentrations of flavopiridol (63 – 500nM) or DMSO vehicle as represented by / PI uptake. (c) Myc lymphoma cell line #20 (Bim null) was cultured for 24 hours in vitro Exponentially growing Eμ- (63 – 500nM) or DMSO vehicle as represented by 0nM. Flow cytometric analysis with varying concentrations of flavopiridol was performed at 24 hours to assess for annexin-V / PI uptake. (d) Myc lymphoma cell line #4242 was stably transduced with an expression vector MSCV- Eμ- Mcl-1-GFP and assessed for proportion of apoptotic cells according to annexin-V / PI uptake following 24 hour incubation with represented concentrations of (e) Myc lymphoma cell line #4242 was stably transduced with an expression vector MSCV-Bcl-2-GFP and assessed for proportion of apoptotic cells flavopiridol. Eμ- according to annexin-V / PI uptake following 24 hour incubation with represented

Dataconcentrations representative of flavopiridol. of mean +/- standard error of the mean of non-viable cells p<0.05 comparing drug to vehicle control (student’s t-test). for three independent experiments. *

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a #4242 b #3391 p53-/- 100 * * 100 * I I * / P / P n n * 50 50 nn ex i nn ex i A A

% %

0 0 3 0 0 9 8 0 6 2 3 7 12 5 25 0 50 0 15 6

Flavopiridol [nM] Flavopiridol [nM]

c #20 Bim-/-

100 * * I / P n 50 nn ex i A

%

0 3 0 6 12 5 25 0 50 0

Flavopiridol [nM] d #4242tmMcl-1 e #4242tmBcl-2

100 100 I * I / P * / P n n 50 50 * nn ex i nn ex i *

A * A

%

* % 0 0 3 0 3 0 6 6 12 5 25 0 50 0 12 5 25 0 50 0

Flavopiridol [nM] Flavopiridol [nM]

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Figure 4.3: A selective CDK4/9 inhibitor induces apoptosis of Eµ-Myc lymphoma in vitro

(a Myc lymphoma cell line #4242 was cultured for 24 hours in vitro with varying concentrations of the CDK4/9 inhibitor A1592668.1 ) Exponentially growing Eμ- (16 – 125nM) or DMSO vehicle as represented by 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake. (b) Exponentially Myc lymphoma cell line #3391 was cultured for 24 hours in vitro with varying concentrations of A1592668.1 (16 – 125nM) or DMSO vehicle growing p53 null Eμ- as represented by 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake. (c) Myc lymphoma cell line #20 was cultured for 24 hours in vitro with varying concentrations Exponentially growing Eμ- of A1592668.1 (16 – 125nM) or DMSO vehicle as represented by 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake. (d) Myc lymphoma cell line #30 was cultured for 24 hours in vitro with varying concentrations of A1592668.1 (16 – 125nM) or DMSO Exponentially growing Eμ- vehicle as represented by 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake. (e) Myc lymphoma cell line #4242 was stably transduced with an expression vector MSCV-Mcl-1-GFP and assessed for Eμ- proportion of apoptotic cells according to annexin-V / PI uptake following 24 hour incubation with represented concentrations of A1592668.1. (f) Myc lymphoma cell line #4242 was stably transduced with an expression vector MSCV-Bcl-2-GFP Eμ- and assessed for proportion of apoptotic cells according to annexin-V / PI uptake following 24 hour incubation with represented concentrations of A1592668.1.

Data representative of mean +/- standard error of the mean of non-viable cells p<0.05 comparing drug to vehicle control (student’s t-test). for three independent experiments. *

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a #4242 b #3391 p53-/- 100 * * 100 * * I * I * / P / P n n * 50 50 nn ex i nn ex i A A

% %

0 0 6 2 3 0 6 2 3 0 1 3 6 1 3 6 12 5 12 5

A1592668.1 [nM] A1592668.1 [nM]

# -/- # -/- c 20 Bim d 30 Bmf

100 * * 100 * I I * / P / P n n * 50 50 * nn ex i nn ex i * A A

% %

0 0 6 2 3 0 6 2 3 0 1 3 6 1 3 6 12 5 12 5

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100 100 I * I / P / P n * n 50 50 nn ex i * nn ex i * A A

% %

0 0 6 2 3 0 6 2 3 0 1 3 6 1 3 6 12 5 12 5

A1592668.1 [nM] A1592668.1 [nM]

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Figure 4.4: The selective CDK4/6 inhibitor palbociclib does not induce apoptosis of Eµ-Myc lymphoma in vitro

(a Myc lymphoma cell line #4242 was cultured for 24 hours in vitro ) Exponentially growing Eμ- (4 – 500nM), the CDK4/6 inhibitor palbociclib (4 – 500nM) or DMSO vehicle as with varying concentrations of dinaciclib (4 – 500nM), flavopiridol represented by 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake. (b) Treated lymphoma cells from the same experiment were prepared for cell cycle analysis using the Nicoletti protocol. Flow cytometry plots depict nuclear DNA content as assessed by PI uptake for lymphoma cells exposed to representative concentrations of palbociclib. (c) Cigar plots for the same experiment showing the proportion of cells in each phase of the cell cycle for representative dinaciclib or palbociclib concentrations.

Data representative of mean +/- standard error of the mean for three independent experiments.

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a #4242

100 I / P n 50 nn ex i A

%

0 6 2 3 6 2 3 6 2 3 0 4 8 0 4 8 0 4 8 1 3 6 1 3 6 1 3 6 12 5 25 0 50 0 12 5 25 0 50 0 12 5 25 0 50 0

Dinaciclib [nM] Flavopiridol [nM] Palbociclib [nM]

b DMSO (0nM) 32nM 500nM

PI

c #4242 G2-M 100 S G1

ce ll s SubG1 l

a 50 To t

%

0 6 6 2 3 0 4 4 8 1 1 3 6 12 5 25 0 50 0 Dinaciclib [nM] Palbociclib [nM]

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Figure 4.5: The selective CDK9 inhibitor AZ-CDK9 induces apoptosis of Eµ-Myc lymphoma in vitro

(a Myc lymphoma cell line #4242 was stably transduced with expression vectors MSCV-Mcl-1-GFP, MSCV-Bcl-2-GFP or vector control (MSCV-GFP, denoted as ) Eμ- # Myc lymphoma cell line #3391 were contemporaneously assessed for proportion 4242) and each of these exponentially growing derived cell lines and p53 null Eμ- of apoptotic cells according to annexin-V / PI uptake following 24 hour incubation with represented concentrations of AZ-CDK9. (b Myc lymphoma cell line #4242 was stably transduced with expression vectors encoding homo sapiens ) Eμ- proteins MSCV-hMCL-1-GFP, MSCV-hBCL-2-GFP, MSCV-hBCL-XL-GFP or vector control (MSCV-GFP, denoted as #4242) and each of these exponentially growing derived cell lines were contemporaneously assessed for proportion of apoptotic cells according to annexin-V / PI uptake following 24 hour incubation with represented concentrations of AZ-CDK9. (c Myc lymphoma cell line #4242 was stably transduced with expression vectors encoding homo sapiens proteins MSCV- ) Eμ- hA1-GFP, MSCV-hBCL-W-GFP or vector control (MSCV-GFP, denoted as #4242) and each of these exponentially growing derived cell lines were contemporaneously assessed for proportion of apoptotic cells according to annexin-V / PI uptake following 24 hour incubation with represented concentrations of AZ-CDK9.

Data representative of mean +/- standard error of the mean of non-viable cells for p<0.05 comparing equal drug concentrations between genotypes (2-way ANOVA). three independent experiments. *

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a #4242 #4242tmMcl-1 #4242tmBcl-2 #3391 p53-/- 100 I * * * * * * / P n 50 nn ex i A

%

0 2 3 2 3 2 3 2 3 0 0 0 0 3 6 3 6 3 6 3 6 12 5 12 5 12 5 12 5

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# # # # b 4242 4242thMCL-1 4242thBCL-2 4242thBCL-XL

100 I

/ P * * * * * * * * * n 50 nn ex i A

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0 2 3 2 3 2 3 2 3 0 0 0 0 6 6 6 6 3 3 3 3 12 5 12 5 12 5 12 5

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#4242 #4242thA1 #4242thBCL-W c 100 I / P

n * * * * * * 50 nn ex i A

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0 2 3 2 3 2 3 0 0 0 3 6 3 6 3 6 12 5 12 5 12 5

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Figure 4.6: Pharmacologic inhibition of CDK9 is associated with cytostasis of apoptosis-protected Eµ-Myc lymphoma in vitro

(a) ‘ Myc lymphoma overexpressing Bcl-2 (#4242tmBcl-2) was incubated with various concentrations of A1592668.1 or DMSO vehicle for 24 Apoptosis-protected’ Eμ- hours prior to preparation of cells using the Nicoletti protocol. Cigar plots depict the proportion of cells in each phase of the cell cycle as assessed by nuclear DNA content measured by PI uptake for representative A1592668.1 concentrations. (b) Myc lymphoma overexpressing Bcl-2 (#4242tmBcl-2) was incubated with various concentrations of AZ-CDK9 or DMSO vehicle for 24 Apoptosis-protected Eμ- hours prior to preparation of cells using the Nicoletti protocol. Cigar plots depict the proportion of cells in each phase of the cell cycle as assessed by nuclear DNA content measured by PI uptake for representative AZ-CDK9 concentrations.

Data representative of mean +/- standard error of the mean for three independent

(2-way ANOVA). experiments. *Denotes significant difference compared to DMSO vehicle control

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a #4242tmBcl-2 G2-M 100 S * * G1 * * SubG1

ce ll s * * l

a 50 To t

%

0 6 2 3 0 8 1 3 6 12 5 25 0 50 0 100 0 A1592668.1 [nM]

b #4242tmBcl-2 G2-M 100 S G1 * * * * SubG1 ce ll s l

a 50 To t

% * * * 0 6 2 3 0 8 1 3 6 12 5 25 0 50 0 100 0 AZ-CDK9 [nM]

133 Chapter 4 induced cytostasis when intrinsic apoptosis was blocked, with a relative reduction in the proportion of cells in S-phase of the cell cycle. Despite protection conferred by Bcl-2 overexpression, higher concentrations of AZ-CDK9 were associated with a relatively increased proportion of cells in the sub-G1 gate (apoptotic) relative to DMSO vehicle control, as opposed to A1592668.1 treatment where increasing concentrations were associated with cytostasis without apoptosis.

Myc lymphoma to human lymphoma, the two CDK9-inhibitors, A1592668.1 and AZ-CDK9, were assessed To confirm relevance of observed apoptosis from Eµ- against a suite of MYC-translocated Burkitt lymphoma and multiple myeloma cell lines using in vitro apoptosis assays (Figure 4.7). Consistent with Eµ-Myc data, potent induction of apoptosis was observed at low nanomolar concentrations for both inhibitors against Burkitt and myeloma cell lines upon 24 hour incubation.

4.2.2 CDK9-inhibitor treatment of Eµ-Myc lymphoma leads to inhibition of RNA polymerase II activation and reduced Mcl-1 expression

In order to support the hypothesis of CDK9 inhibition as the critical mechanism transcriptional effects of the novel CDK9 inhibitors were performed as to those of dinaciclib-mediated apoptosis induction, similar profiling of the signalling and experiments shown with dinaciclib in chapter three. As with dinaciclib, short incubations of human IG-cMYC-translocated lymphoma cell lines with AZ-CDK9 MCL-1 mRNA as assessed by RT-PCR (Figure 4.8a). However, in contrast to positive controls of dinaciclib were sufficient to effect a significant reduction in and bromodomain inhibition with JQ1 for potent suppression of cMYC, AZ-CDK9 caused a relative increase in cMYC mRNA at this short timepoint (Figure 4.8a). When the same experiment was performed to assess the transcriptional effects of A1592668.1, treatment was associated with a reduction in cMYC mRNA expression (Figure 4.8b) although this experiment was only performed once and would need to be repeated.

Signalling effects of the CDK9 inhibitors were next assessed using Eµ-Myc lymphoma cell lysates with Western blotting and immunostaining. As was seen with dinaciclib in chapter three, short treatments of lymphoma cells with each

(Figure 4.9a,b CDK9 inhibitor were associated with a significant reduction in pRpb1(Ser2) with dinaciclib, this effect was overcome when cells with forced overexpression ). A significant reduction in Mcl-1 protein was also seen, and as of Mcl-1 were treated (Figure 4.9a L protein expression was observed (Figure 4.9b). The same effects of AZ-CDK9 on ). No significant reduction in Bcl-2 or Bcl-x

134 Chapter 4 anti-apoptotic protein expression were also observed upon three-hour incubation of the human Burkitt lymphoma line BL-41 (Figure 4.9c). These results are consistent with the hypothesised dinaciclib-mediated inhibition of CDK9 leading to suppression of Pol II activation and subsequent reduction of MCL-1 to induce apoptosis.

4.2.3 CDK9-inhibitor therapy is associated with prolonged survival of tumour-bearing mice in vivo

Having shown similar effects to dinaciclib in vitro, A1592668.1 was next assessed for in vivo activity. Pharmaceutical industry collaborators provided data showing a maximum tolerated dose (MTD) of 5mg/kg mouse weight. An initial pilot in vivo MTD of A1592668.1 5mg/kg administered three times weekly by oral gavage in vehicle comprising 73% hydroxypropylmethylcellulose (HPMC), 20% PEG400, 5% Tween and 2% DMSO in non-tumour bearing C57BL/6 mice showed tolerability of Figure 4.10a). Given that future combination studies may be performed in conjunction with other this dose with no significant toxicity including weight loss ( targeted therapeutics, subsequent studies were performed using a dose of 3mg/ kg as is used by our industry collaborator when in combination with other drugs.

Having demonstrated tolerability, in vivo assays were performed to assess whether A1592668.1 could induce the same effects on Pol II and Mcl-1 signalling in tumour- bearing mice as were seen in vitro. C57BL/6 mice were transplanted with Eµ- Myc lymphoma and left untreated to establish bulky lymph nodes. Following a single 3mg/kg dose of A1592668.1 or vehicle (73% HPMC), lymph node-derived reduction of Mcl-1 protein expression (Figure 4.10b cell lysates were obtained and confirmed inhibition of Pol II activation and of A1592668.1 in vivo as to that observed in vitro. Nuclear DNA content analysis ), confirming the same effect using a Nicoletti protocol also showed an increased proportion of apoptotic cells with fragmented DNA following exposure to A1592668.1 when compared with vehicle treatment (Figure 4.10b inhibitor is able to induce apoptosis in vivo, with the same signalling effects as ). These findings confirm that this selective CDK9 were seen with dinaciclib in chapter three.

Finally, a study was performed to assess for a possible survival advantage conferred by A1592668.1 treatment to syngeneic mice transplanted with Eµ-Myc lymphoma (Figure 4.11a disease progression as shown by number of lymphoma cells in leukaemic phase ). Three weeks of therapy was observed to significantly abrogate Figure 4.11b,c). at day 12, with no significant haematological toxicity observed ( 135 Chapter 4

Figure 4.7: Pharmacologic inhibition of CDK9 is associated with apoptosis of human IG-MYC-translocated lymphoma and myeloma cell lines in vitro

(a) Exponentially growing IG-cMYC-translocated human Burkitt lymphoma (grey bars) and multiple myeloma (white bars) cell lines were cultured for 24 hours in vitro with varying concentrations of AZ-CDK9 (32 – 500nM) or DMSO vehicle as represented by 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake. (b) Exponentially growing IG-cMYC-translocated human Burkitt lymphoma cell lines were cultured for 24 hours in vitro with varying concentrations of A1592668.1 (16 – 64nM) or DMSO vehicle as represented by 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake. (c) Exponentially growing IG-MYC-translocated human multiple myeloma cell lines (JJN3 cMYC and U266 L-Myc) were cultured for 24 hours in vitro with varying concentrations of A1592668.1 (16 – 64nM) or DMSO vehicle as represented by 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake.

Data representative of mean +/- standard error of the mean for three independent p<0.05 comparing drug concentrations to vehicle within each individual cell line (2-way ANOVA). experiments. *

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a BL-41 Ramos Namalwa OPM2 H929 100 * * * * * * * I * * * * / P n * * 50 nn ex i A

%

0 2 3 2 3 2 3 2 3 2 3 0 0 0 0 0 3 6 3 6 3 6 3 6 3 6 12 5 25 0 50 0 12 5 25 0 50 0 12 5 25 0 50 0 12 5 25 0 50 0 12 5 25 0 50 0

AZ-CDK9 [nM]

b BL-41 Ramos c JJN3 U266

100 * * * * 100 * *

I I *

/ P * * / P * n n 50 50 * nn ex i nn ex i A A

% %

0 0 6 2 4 6 2 4 6 2 4 6 2 4 0 0 0 0 1 3 6 1 3 6 1 3 6 1 3 6

A1592668.1 [nM] A1592668.1 [nM]

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Figure 4.8: Pharmacologic inhibition of CDK9 is associated with repression of MCL-1 transcription in vitro

(a) Human IG-cMYC-translocated Burkitt lymphoma cell lines were incubated for two hours in vitro with DMSO vehicle, AZ-CDK9 60nM, dinaciclib 20nM or JQ1 500nM prior to harvesting of cell pellets and RNA extraction. Quantitative RT-PCR was performed using primer sets for MCL-1 (left) and cMYC (right) and normalized to the L32 p p p p<0.0001 (student’s t-test comparing DMSO vehicle to represented therapy). housekeeping gene. * <0.05, ** <0.005, *** <0.0005, ****

Data representative of mean +/- standard error of the mean for three independent experiments.

(b) Human IG-cMYC-translocated Burkitt lymphoma cell lines were incubated for two hours in vitro with DMSO vehicle, A1592668.1 100nM, dinaciclib 20nM or JQ1 500nM prior to harvesting of cell pellets and RNA extraction. Quantitative RT-PCR was performed using primer sets for cMYC normalized to the L32 housekeeping gene.

Data obtained from a single experiment.

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a MCL-1 cMYC

BL-41 Ramos BL-41 Ramos ) **** *** ) *** *** * ** * * MS O 1.0 MS O 2.0 D D ( ( 1.5 eve l eve l 0.5 1.0 mRNA l mRNA l 0.5 ve ve i i t t a 0.0 a 0.0 9 9 9 9 l l e e JQ 1 JQ 1 R R DMSO DMSO DMSO DMSO AZ-CDK AZ-CDK AZ-CDK AZ-CDK Dinaciclib Dinaciclib Dinaciclib Dinaciclib

b cMYC

) BL-41 Ramos

MS O 1.0 D ( eve l 0.5 mRNA l ve i t

a 0.0 l 1 1 e R JQ 1 JQ 1 DMSO DMSO Dinaciclib Dinaciclib A1592668. A1592668.

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Figure 4.9: CDK9-inhibitor treatment is associated with reduced Pol II activation and Mcl-1 protein expression in MYC-driven lymphoma cells

(a) Myc lymphoma cells (# Myc lymphoma cells overexpressing Mcl-1 (#4242tmMcl-1) were cultured in the presence of DMSO Eμ- 4242) and ‘apoptosis-protected’ Eμ- vehicle or A1592668.1 100nM for three hours prior to Western blotting. Protein gel using SDS-PAGE prior to immunoblotting for RNA polymerase II, subunit B1 extracted from whole cell lysates (10μg) was separated on gradient polyacrylamide phosphorylation at Serine 2/5 (pRpb1Ser2/5), Mcl-1 and HSP90 loading control. (b) Myc lymphoma cells (#4242) were cultured in the presence of DMSO vehicle or AZ-CDK9 60nM for three hours prior to Western blotting. Protein extracted from Eμ-

PAGE prior to immunoblotting for RNA polymerase II, subunit B1 phosphorylation whole cell lysates (10μg) was separated on gradient polyacrylamide gel using SDS- at Serine 2 (pRpb1Ser2), Bcl-xL and Tubulin loading control. Protein extracted from gel using SDS-PAGE prior to immunoblotting for RNA polymerase II, subunit B1 whole cell lysates (10μg) was separated on a second gradient polyacrylamide phosphorylation at Serine 5 (pRpb1Ser5), Mcl-1, Bcl-2 and Tubulin loading control. (c) BL-41 human Burkitt lymphoma cells were cultured in the presence of DMSO vehicle or AZ-CDK9 60nM for three hours prior to Western blotting. Protein gel using SDS-PAGE prior to immunoblotting for Bcl-xL and Tubulin loading extracted from whole cell lysates (10μg) was separated on gradient polyacrylamide second gradient polyacrylamide gel using SDS-PAGE prior to immunoblotting for control. Protein extracted from whole cell lysates (10μg) was separated on a Mcl-1, Bcl-2 and Tubulin loading control.

Data representative of experiment performed in triplicate.

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a #4242 #4242tmMcl-1 DMSO A1592668.1 DMSO A1592668.1

pRpb1Ser2/5 250 kDa

Mcl-1 40 kDa

HSP90 90 kDa

b #4242 c BL-41 DMSO AZ-CDK9 DMSO AZ-CDK9 pRpb1Ser2 250 kDa Bcl-xL 30 kDa

Bcl-xL 30 kDa Tubulin 52 kDa

Tubulin 52 kDa Mcl-1 40 kDa

30 kDa pRpb1Ser5 250 kDa Bcl-2

Tubulin 52 kDa Mcl-1 40 kDa

Bcl-2 30 kDa Tubulin 52 kDa

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Figure 4.10: CDK9 inhibitor therapy is associated with reduced Pol II activation and Mcl-1 protein expression in vivo

(a) Non-tumour bearing C57BL/6 mice were dosed with vehicle 73% hydroxypropylmethylcellulose (HPMC) or A1592668.1 3mg/kg, three times weekly by oral gavage for three weeks. Weights of individual mice at completion of

(b) Non-irradiated C57BL/6 recipient mice were transplanted by tail vein injection three weeks of therapy relative to baseline weights are shown. NS not significant. with 1 x 105 Myc #4242 lymphoma cells harvested from lymph nodes of a syngeneic mouse. After allowing 12 days to establish bulky lymph nodal disease, Eμ- mice received a single dose of vehicle 73% HPMC or A1592668.1 3mg/kg, prior to and prepared as a single cell suspension from which a proportion were prepared sacrifice three hours later. Lymph nodes were harvested from independent mice according to Nicoletti protocol for assessment of cell cycle effects according to left). The other proportion of cells underwent cell lysis and protein extraction. nuclear DNA content as represented by flow cytometric analysis of PI uptake (lower polyacrylamide gel using SDS-PAGE prior to immunoblotting for pRpb1Ser2/5, Mcl-1 Protein extracted from whole cell lysates (10μg) was separated on gradient and HSP90 loading control. Each lane represents protein from cell lysate derived from lymph nodes of a single mouse.

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a NS

) 150 e

ase li n 100 b ( gh t

i 50 e W

% 0 1 Vehicl e b A1592668. Eµ-Myc 1x105 cells

Day 12

73% HPMC vehicle vs. A1592668.1 3mg/kg by oral gavage

#4242 G2-M 100 S G1 SubG1 ce ll s l a 50 Vehicle A1592668.1 To t

% pRpb1Ser2/5 250 kDa

0 1 Mcl-1 40 kDa

HSP90 90 kDa Vehicl e A1592668.

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Figure 4.11: CDK9 inhibitor therapy is associated delayed disease progression and prolongation of overall survival in vivo

(a) Non-irradiated C57BL/6 recipient mice were transplanted by tail vein injection with 1 x 105 Myc #4242 lymphoma cells harvested from lymph nodes of a syngeneic mouse. Therapy was commenced three days later with 73% HPMC Eμ- vehicle or A1592668.1 3mg/kg, administered three times weekly by oral gavage. (b) Mice from the same experiment were bled at day 12 post-transplantation and assessed by full blood examination for effects on haemoglobin, neutrophil count (c) Absolute number of lymphocytes in peripheral blood (lymphoblasts in leukaemic phase) from the same full blood and platelet count. NS, not significant. p<0.0001. (d) Kaplan-Meier survival curve of mice from the Myc lymphoma #4242 experiment. Median survival for vehicle-treated examination. *** mice (n=10) was 13 days and for A1592668.1-treated mice (n=10) was 24 days same Eμ- (p<0.0001, Log-rank test).

Data representative of mean +/- standard error of the mean for representative treatment group.

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a Eµ-Myc 1x105 cells

Day 3 Day 21

73% HPMC vehicle vs. A1592668.1 3mg/kg three times weekly by oral gavage

b NS NS

/L ) NS 9 /L )

g/L ) 6 200 9 600 ( x1 0 (

4 x1 0

( 400

100 s t e oglobin

2 l 200 ophil s e r t t a u l ae m 0 0 0 1 1 e 1 P H N Vehicl e Vehicl e Vehicl e A1592668. A1592668. A1592668.

c d #4242 /L ) 9 A1592668.1 80 *** 100

x1 0 Vehicle va l ( 60 rv i es 50 40 u S

20 % 0 0 1 0 10 20 30 40 ym pho cy t L Days post transplantation Vehicl e A1592668.

145 Chapter 4

was observed for mice treated with A1592668.1 when compared to those treated Following completion of three weeks of therapy, a significant survival advantage with HPMC vehicle control (Figure 4.11d). These results again provide evidence for the use of targeted CDK9 inhibition for the treatment of aggressive MYC-driven lymphoma.

4.2.4 CDK9-inhibitor-mediated repression of Mcl-1 and inhibition of Bcl-2 display additivity in vitro and synergise in vivo in MYC- translocated lymphoid malignancy

Given the recent success of preclinical and clinical studies of the Bcl-2 inhibitor ABT-199 (venetoclax) in certain B-cell neoplasms(Souers et al. 2013; Roberts et al. 2016), it was logical to next assess whether Mcl-1 repression mediated through CDK9 inhibition would synergise with antagonism of anti-apoptotic Bcl-2 to effect more complete apoptosis induction. Previous published data has shown relative resistance of Eµ-Myc lymphomas to Bcl-2 inhibition with ABT-199 as monotherapy (Mason et al. 2008).

Despite a quoted IC50 of <0.01nM for BCL-2 inhibition (Souers et al. 2013), in vitro apoptosis assays performed using several independent spontaneously- arising and derived Eµ-Myc lymphomas revealed an LD50 predominantly in the micromolar range (Figure 4.12a-c). While overexpression of murine Mcl-1 was found to be protective against ABT-199-induced apoptosis (Figure 4.12d), forced overexpression of murine Bcl-2 was observed to reprogram the lymphoma cells to markedly increase their sensitivity to Bcl-2 inhibition and reduce the LD50 to low nanomolar concentrations (Figure 4.12e).

Checkerboard synergy apoptosis assay experiments were performed with varying concentrations of dinaciclib or A1592668.1 in combination with ABT-199 using Eµ-Myc lymphoma #4242, and did not demonstrate any appreciable synergy (Figure 4.13a,b). Indeed, statistical analysis for synergy using the Chou-Talalay Combination Index method (Chou 2006) revealed mild antagonism of these agents when combined in vitro at concentrations below the respective LD50 of each single agent (Figure 4.13c). Furthermore, the addition of Bcl-2 inhibition with ABT-199 was not able to overcome the protection from CDK9 inhibitor-mediated apoptosis conferred by overexpression of Mcl-1 (Figure 4.13d,e).

Despite a lack of in vitro in vivo as our group and others have observed unexpected activity or lack of activity synergy, the combination was also assessed for efficacy

146 Chapter 4 discordant between in vitro and in vivo studies across a range of malignancies and therapeutic classes attributed to immunological mechanisms or upregulation of compensatory signalling pathways (West et al. 2016, Haynes, unpublished data). Mice were transplanted with Eµ-Myc lymphoma three days prior to three weeks of therapy with dinaciclib 20mg/kg and ABT-199 50mg/kg, A1592668.1 3mg/kg and ABT-199 50mg/kg or appropriate single therapy or vehicle treatment controls (Figure 4.14a). Reduced-dose regimens (dinaciclib 20mg/kg and ABT-199 50mg/ Myc experiment described in the next paragraph. Strikingly, monotherapy with ABT-199 was associated with disease kg) were used due to toxicities in the Vκ* acceleration when compared to vehicle controls (Figure 4.14b). This observation has not previously been reported by other groups despite their reporting of a lack with monotherapy or combination therapy were associated with a statistically of therapeutic efficacy (Mason et al. 2008). In contrast, all other therapy arms Figure 4.14b). Furthermore, there was a trend toward improved overall survival from the significant survival benefit when compared to the vehicle group ( addition of ABT-199 to CDK9 inhibition with A1592668.1 when compared to the

A1592668.1 single arm control, though this did not reach statistical significance. No significant difference in overall survival was observed between the dinaciclib monotherapy and dinaciclib/ABT-199 combination arms, though the significant potential synergy in this instance. single agent activity of the former may confer difficulty when assessing for

Myc murine Myc-driven multiple myeloma model was also assessed for activity of CDK9 inhibition and Bcl-2 antagonism combination therapy. The aggressive Vκ* This model was chosen as it is a relevant and transplantable Myc-driven model, it has previously shown utility for assessment of therapeutics in vivo, and therapy with CDK9 inhibition or Bcl-2 antagonism have each shown activity against myeloma in the pre-clinical or clinical settings (Matthews et al. 2013; Gojo et al. 2002; Kumar et al. 2014). Tumour was transplanted into sublethally-irradiated syngeneic C57BL/6 mice and engraftment assessed by serum protein electrophoresis (SPEP). ensure the approximate median and range of monoclonal protein (M-protein) was Following confirmation of engraftment, mice were assigned to treatment arms to comparable prior to commencement of three weeks of therapy with dinaciclib 30mg/kg and ABT-199 100mg/kg, A1592668.1 3mg/kg and ABT-199 100mg/kg or appropriate single therapy or vehicle treatment controls (Figure 4.15a).

Repeat SPEP analysis following one week of therapy (normalised to baseline SPEP) indicated progression of this aggressive myeloma model for all treatment arms (Figures 4.15b,c

). However, despite a lack of statistical significance, there 147 Chapter 4

Figure 4.12: Pharmacologic inhibition of Bcl-2 does not induce apoptosis of Eμ-Myc lymphoma at on-target concentrations in vitro

(a) Myc lymphoma cell line #4242 was cultured for 24 hours in vitro with varying concentrations of ABT-199 (156 – 2500nM) or DMSO Exponentially growing Eμ- vehicle as represented by 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake. (b) Exponentially growing p53 null Myc lymphoma cell line #3391 was cultured for 24 hours in vitro with varying concentrations of ABT-199 (1000 – 4000nM) or DMSO vehicle as represented by Eμ- 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake. (c) Myc lymphoma cell line #20 was cultured for 24 hours in vitro with varying concentrations of ABT-199 (312 – 5000nM) or Exponentially growing Eμ- DMSO vehicle as represented by 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake. (d) Myc lymphoma cell line #4242 was stably transduced with an expression vector Exponentially growing Eμ- MSCV-Mcl-1-GFP and incubated with varying concentrations of ABT-199 (1250 – 10000nM) or DMSO vehicle as represented by 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake. (e) Exponentially Myc lymphoma cell line #4242 was stably transduced with an expression vector MSCV-Bcl-2-GFP and incubated with varying concentrations of growing Eμ- ABT-199 (8 – 250nM) or DMSO vehicle as represented by 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake.

Data representative of mean +/- standard error of the mean for three independent experiments, except for the 4000nM treatment of #3391 cells which was performed p<0.05 comparing drug to vehicle control (student’s t-test). in duplicate. *

148 Chapter 4

a #4242 b #3391 p53-/- 100 * * 100 I I * / P / P n n 50 50 nn ex i nn ex i A A

% %

0 0 0 0 15 6 31 2 62 5 100 0 200 0 400 0 125 0 250 0 ABT-199 [nM] ABT-199 [nM]

c #20 Bim-/- d #4242tmMcl-1 100 * 100 * * I I * / P / P n n * 50 50 nn ex i nn ex i

* A A

% %

0 0 0 0 31 2 62 5 125 0 250 0 500 0 125 0 250 0 500 0 1000 0 ABT-199 [nM] ABT-199 [nM]

e #4242tmBcl-2 100

I * *

/ P * n * 50 nn ex i

A *

%

0 6 2 3 0 8 1 3 6 12 5 25 0 ABT-199 [nM]

149 Chapter 4

Figure 4.13: Pharmacologic inhibition of Bcl-2 does not synergise with CDK9 inhibition to induce apoptosis of Eμ-Myc lymphoma in vitro

(a) Myc lymphoma cell line #4242 was cultured for 24 hours in vitro with varying concentrations of ABT-199 (0 – 1000nM) in Exponentially growing Eμ- combination with varying concentrations of dinaciclib (4 – 16nM) or DMSO vehicle as represented by 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake. (b) Myc lymphoma cell line #4242 was cultured for 24 hours in vitro with varying concentrations of ABT- Exponentially growing Eμ- 199 (0 – 1000nM) in combination with varying concentrations of A1592668.1 (16 – 63nM) or DMSO vehicle as represented by 0nM. Flow cytometric analysis was performed at 24 hours to assess for annexin-V / PI uptake. (c) Statistical analysis of the apoptosis observed in (a) above using the Chou-Talalay Combination Index method. Figure denotes the combination index (CI) graphed against the fraction affected (Fa), where CI < 1 indicates synergy and CI > 1 indicates antagonism. Table denotes fraction affected (Effect) and CI for represented dose combinations. Values used for statistical analyses were the mean proportion of apoptotic cells for each dosing combination from triplicate experiment shown in a. (d) Myc lymphoma cell line #4242 was stably transduced with an expression vector MSCV-Mcl-1-GFP and cultured for 24 hours in vitro with Exponentially growing Eμ- varying concentrations of ABT-199 (0 – 1000nM) in combination with varying concentrations of dinaciclib (4 – 16nM) or DMSO vehicle as represented by (e) The same experiment was performed with varying concentrations of ABT-199 (0 – 1000nM) 0nM, prior to flow cytometric analysis for annexin-V / PI uptake. in combination with varying concentrations of A1592668.1 (16 – 63nM) or DMSO

PI uptake. vehicle as represented by 0nM, prior to flow cytometric analysis for annexin-V /

Data representative of mean +/- standard error of the mean for three independent experiments.

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a #4242 b #4242

ABT-199 [nM] ABT-199 [nM] 0 500 1000 0 500 1000 100 100 I I / P / P n n 50 50 nn ex i nn ex i A A

% %

0 0 6 6 6 0 4 8 0 4 8 0 4 8 6 2 3 6 2 3 6 2 3 0 0 0 1 1 1 1 3 6 1 3 6 1 3 6 Dinaciclib [nM] A1592668.1 [nM] c 2

Dinaciclib ABT-199 Effect CI [nM] [nM]

CI 1 4 500 0.453 1.87245 4 1000 0.62 1.37522 8 500 0.687 1.07093 8 1000 0.787 0.82181

0 0.5 1 Fa d #4242tmMcl-1 e #4242tmMcl-1

ABT-199 [nM] ABT-199 [nM] 0 500 1000 0 500 1000 100 100 I I / P / P n n 50 50 nn ex i nn ex i A A

% %

0 0 6 6 6 0 4 8 0 4 8 0 4 8 6 2 3 6 2 3 6 2 3 0 0 0 1 1 1 1 3 6 1 3 6 1 3 6 Dinaciclib [nM] A1592668.1 [nM]

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Figure 4.14: Pharmacologic inhibition of Bcl-2 in combination with CDK9 inhibition is associated with prolonged survival of Eµ-Myc tumour-bearing mice in vivo

(a) C57BL/6 mice were transplanted with Eµ-Myc lymphoma #4242 harvested from a syngeneic mouse. Three days later, three weeks of therapy was commenced with 73% HPMC vehicle (oral gavage three times per week) and phosal vehicle (oral gavage daily), 20% HPBCD vehicle (intraperitoneal injection twice weekly) and phosal vehicle (oral gavage daily), A1592668.1 3mg/kg (oral gavage three times per week), ABT-199 50mg/kg (oral gavage daily), dinaciclib 20mg/kg (intraperitoneal injection twice weekly), A1592668.1 3mg/kg (oral gavage three times per week) and ABT-199 50mg/kg (oral gavage daily), or dinaciclib 20mg/ kg (intraperitoneal injection twice weekly) and ABT-199 50mg/kg (oral gavage daily). (b) Kaplan-Meier survival curves for mice in all treatment arms. The two vehicle arms were combined and are presented as ‘Vehicle’. Grey shading denotes period of therapy. Median OS was 17 days (Vehicle, n=12), 30.5 days (A1592668.1, n=6), 13 days (ABT-199, n=6), 41 days (A1592668.1 & ABT-199, n=10), 45 days (dinaciclib, n=6), 41 days (dinaciclib & ABT-199, n=10). Compared with vehicle group, p<0.001 (ABT-199), p<0.005 (dinaciclib), p<0.005 (A1592668.1), p<0.0001 (dinaciclib & ABT-199), p<0.0001 (A1592668.1 & ABT-199), Log-rank test. p=0.053 comparing A1592668.1 to A1592668.1 & ABT-199, (Log-rank test).

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a Eµ-Myc Three weeks 1x105 cells

• 73% HPMC & Phosal vehicle • 20% HPBCD & Phosal vehicle • A1592668.1 3mg/kg • Dinaciclib 20mg/kg • ABT-199 500mg/kg • A1592668.1 & ABT-199 • Dinaciclib & ABT-199

b

100 Vehicle Dinaciclib va l A1592668.1 rv i 50 u ABT199 S

% Dinaciclib & ABT199 A1592668.1 & ABT199 0 0 20 40 60 Days post transplantation

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Figure 4.15: Combination pharmacologic inhibition of Bcl-2 and CDK9 is associated with prolonged survival of Vκ*Myc tumour-bearing mice in vivo

(a) C57BL/6 mice were sublethally irradiated with two fractions of 3.3Gy, one day Myc clone #4929 splenocytes. Mice were bled six prior to transplantation of Vκ* quantitated baseline percentage monoclonal protein pre-treatment. Mice were weeks later and serum protein electrophoresis (SPEP) confirmed engraftment and allocated treatment groups to have comparable mean and range of paraproteins for each group at baseline. Three weeks of therapy was commenced at day 45 post- transplantation with 73% HPMC vehicle (oral gavage three times per week) and phosal vehicle (oral gavage daily), 20% HPBCD vehicle (IP injection twice weekly) and phosal vehicle (oral gavage daily), A1592668.1 3mg/kg (oral gavage three times per week), ABT-199 100mg/kg (oral gavage daily), dinaciclib 30mg/kg (IP injection twice weekly), A1592668.1 3mg/kg (oral gavage three times per week) and ABT-199 100mg/kg (oral gavage daily), or dinaciclib 30mg/kg (IP injection twice weekly) and ABT-199 100mg/kg (oral gavage daily).

(b) Mice were bled at day 52 post-transplantation (day seven post commencement of therapy) and percentage monoclonal protein was analysed by SPEP. Fold change of percent monoclonal protein (normalized to baseline) shown for each treatment p p=0.03 (student’s t-test). group. NS not significant, NS* not significant =0.055, ** (c) The same SPEP data as b shown for vehicle (HPMC & phosal) or A1592668.1 & ABT-199, comparing the day seven fold-change data to baseline (arbitrarily

p=0.014 HPMC & phosal, p=0.033 A1592668.1 & ABT-199 (student’s defined as 1) to assess probability of progression within respective treatment t-test). group. **

(d) Kaplan-Meier survival curves for mice in all treatment arms, showing A1592668.1 therapy groups and controls (left) and dinaciclib therapy groups and controls (right) from the same experiment. Grey shading denotes period of therapy. Median OS 71 days (HPMC & phosal, n=6), 79 days (A1592668.1, n=6), 67 days (ABT-199, n=6), 88 days (A1592668.1 & ABT-199, n=10), 83 days (HPBCD & phosal, n=6), 80 days (dinaciclib, n=6), 57 days (dinaciclib & ABT-199, n=10). p=0.026 comparing HPMC & phosal vehicle to A1592668.1 & ABT-199 combination group (Log-rank test).

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a V *Myc Three weeks 1x105 cells

• 73% HPMC & Phosal vehicle • 20% HPBCD & Phosal vehicle • A1592668.1 3mg/kg • Dinaciclib 30mg/kg • ABT-199 100mg/kg • A1592668.1 & ABT-199 • Dinaciclib & ABT-199 b NS c

NS ) ) 6 ** 6 NS* ** ** ase lin e ase lin e b b 4 4 ( (

ng e ng e a a 2 2 h h c c

Fold 0

Fold 0 l l l 9 -19 9 -19 9 ABT-19 9 AB T AB T

Dinacicli b & &

A1592668. 1 1 1 . . HPMC & Phosa HPMC & Phosa HPBCD & Phosa Dinaciclib & ABT-19 A1592668 A1592668 d

100 HPMC & Phosal 100 HPBCD & Phosal A1592668.1 va l va l Dinaciclib ABT-199 rv i 50 rv i 50 ABT-199 u u S A1592668.1 S Dinaciclib

& ABT-199 & ABT-199 % % 0 0 0 50 100 150 0 50 100 150 Days post transplantation Days post transplantation

155 Chapter 4 was a trend toward abrogated disease progression with the combination of CDK9 inhibition with A1592668.1 or dinaciclib, in combination with Bcl-2 antagonism with ABT-199. An unfortunate observation was of exacerbated gastrointestinal toxicity of dinaciclib when combined with ABT-199. This had not been observed in non-tumour bearing or tumour bearing mice by industry collaborators (data not shown) and it is hypothesised that the toxicity observed may be attributable to therapies in the context of renal impairment complicating myeloma. Two interesting observations were made from this experiment. Firstly, as was seen in the Eµ-Myc experiment, monotherapy with ABT-199 again appeared to accelerate disease progression according to day seven SPEP analysis and was also associated with reduced overall survival. Secondly, consistent with abrogation of disease progression, the combination of A1592668.1 and ABT-199 was associated with Figure 4.15d). Neither dinaciclib nor A1592668.1 monotherapy were able to confer any survival a significant OS advantage compared to vehicle-treated controls ( may indeed confer in vivo synergy against this disease model. advantage. These findings suggest that CDK9 inhibition and Bcl-2 antagonism

4.2.5 Genetic depletion of Cdk9 is detrimental to Eµ-Myc lymphoma

Previous work performed in our laboratory by Ms Adele Baker included use of constitutive short hairpin (sh)RNAs targeting CDK9 in mixed lineage leukaemia (MLL)-translocated acute myeloid leukaemia (AML). In that disease model, silencing of CDK9 mRNA in the MLL-rearranged THP-1 cell line was associated with a significant reduction in colony-forming capacity (Baker et al. 2016). In order to assess whether genetic de-induction of Cdk9 is similarly detrimental to Eµ-Myc lymphoma as was seen in Ms Baker’s AML experiments, and as would be predicted following the observed results of pharmacologic CDK9 inhibition, the next experiments were performed to assess the effects of shRNA against Cdk9 in Eµ-Myc lymphoma. Several candidate targeting hairpins were sourced from commercial suppliers with little suppression of Cdk9 observed upon transduction of target cells (data not shown). During this period, independent research was published highlighting the potential for targeting Cdk9 in MYC-driven hairpins, oligonucleotides were purchased containing the same sequences as used hepatocellular carcinoma (Huang et al. 2014). Due to the efficacy of their targeting in the published studies.

The Cdk9 targeting oligonucleotides were next cloned into an miR-E (Fellmann et al. 2013) tetracycline-inducible vector (REBIR) developed and validated in the

156 Chapter 4 laboratory by Mr Sang-Kyu Kim (Figure 4.16a). This construct enables tracking and inducible expression of dsRED upon doxycycline activation of the tetracycline- of transduced cells with constitutive blue fluorescent protein (BFP) expression, responsive element (TRE) with subsequent transcription of the shRNA targeting Cdk9 (shCdk9) leading to depletion of endogenous Cdk9 mRNA. These inducible shCdk9 constructs and non-silencing scrambled controls (shScrambled) were transduced into Eµ-Myc lymphoma cell line #4242 and assessed for depletion of Cdk9 by immunoblotting of cell lysates with Western blot when exposed to doxycycline. Upon addition of doxycycline, the shCdk9.421 construct was associated with the greatest depletion of Cdk9 protein expression (Figure 4.16b). Concomitant to this, reduction of pRpb1(Ser2/5) was also observed, demonstrating reduction of Pol II activation in the absence of Cdk9 (Figure 4.16b). This phenocopies the observed effects of pharmacologic inhibition of Cdk9. Finally, when transduced #4242 cells were incubated and passaged in the presence of doxycycline, the proportion of dsRED positive cells (‘switching on’ the shCdk9 and depleting Cdk9 protein) was when compared to the other shRNAs (Figure 4.16c seen to rapidly and significantly diminish for those containing the shCdk9.421 pharmacologic studies and implicate Cdk9 as a critical oncogenic requirement of ). These findings support the Eµ-Myc lymphoma.

4.2.6 A boutique CDK RNA-interference screen identifies novel CDKs which are critical to MYC-driven lymphoid malignancy

The previous experiments in this chapter have established CDK9 as a targetable oncogenic requirement of MYC-driven lymphoid malignancy. However, some interesting observed differences in vitro between dinaciclib and selective CDK9 inhibitors, and the more striking in vivo therapeutic may possess synergistic activity against other CDK or non-CDK targets. efficacy of dinaciclib suggests that this In order to assess for the oncogenic requirement of each individual CDK, a boutique RNA-interference (RNAi) screen was performed in collaboration with the Victorian Centre for Functional Genomics (VCFG). Lentiviral constructs targeting each of CDK1 - CDK13 were sourced from the in-house library (VCFG) and virus produced and batched using polyethylenimine (PEI) transfection of HEK293T cells for the following experiments. It should be noted that the majority of hairpins contained within this library are algorithm-generated and had not been previously validated for efficacy of target depletion. A similar screen of targets of immunomodulatory agents and epigenetic therapies was performed in the laboratory contemporaneously by Ms Leonie Cluse. This

157 Chapter 4

Figure 4.16: Genetic depletion of Cdk9 with shRNA phenocopies CDK9 inhibition of Eµ-Myc lymphoma

(a) Schematic representation of the miR-E tetracycline-inducible shRNA.REBIR construct. TRE, tetracycline responsive element; dsRED, Discosoma sp. Red protein; IRES, internal ribosome entry site; rtTA, reverse tetracycline-controlled fluorescent protein; PGK, phosphoglycerate kinase promoter; BFP, blue fluorescent transactivator. (b) The REBIR.shCdk9 constructs and non-silencing control (shScrambled) were cloned into Eµ-Myc #4242 lymphoma cells. Transduced cells with or without the addition of doxycycline 1µg/mL. Protein was extracted 48 were sorted for BFP expression by flow cytometry then exponentially grown polyacrylamide gel using SDS-PAGE prior to immunoblotting for Cdk9 and tubulin hours later from whole cell lysates. Protein (10μg) was separated on gradient loading control (left), and RNA polymerase II, subunit B1 phosphorylation at Serine 2/5 (pRpb1Ser2/5) and tubulin loading control (right). (c) Eµ-Myc #4242 lymphoma cells transduced with shCdk9 constructs or shScrambled were exponentially passaged in competitive culture with non-transduced cells, with or without the addition of doxycycline 1µg/mL. Flow cytometric analysis of dsRED expression was performed 48 hours post-addition of doxycycline (arbitrarily defined as day p<0.001 comparing shCdk9.421 to shScrambled non- 0) and again five days later. Day 5 values are shown normalized to day 0 dsRED silencing control. positive proportion. ***

Data representative of mean +/- standard error of the mean for three independent experiments.

158 Chapter 4

a shCdk9miR-E

TRE dsRED PGK BFP IRES rtTA

b shScrambled shCdk9.421 shScrambled shCdk9.2869 shCdk9.421

Doxycycline - + - + - + Doxycycline - + - +

Cdk9 42 kDa pRpb1Ser2/5 250 kDa

Tubulin 52 kDa Tubulin 52 kDa

c ) shScrambled e 100 shCdk9.421 shCdk9.2869 Base li n ( *** D 50 sR E d

% 0 2 4 6 Time (days)

159 Chapter 4 involved optimisation of the method for lentiviral transduction of target human MYC-driven Burkitt lymphoma and multiple myeloma cell lines. From this optimisation, it was observed that the human IG-cMYC-translocated multiple using the lentiviral system. The same target cell line was used for the lentiviral myeloma cell line, OPM2, was associated with the greatest transduction efficiency RNAi CDK experiments, given that the OPM2 cell line had already been shown to be sensitive to dinaciclib or AZ-CDK9 inhibitor-mediated apoptosis in previous experiments (Figure 3.1, Figure 4.7).

In the initial screen, multiple GFP-tagged hairpins targeting CDK1 to CDK13, and non-silencing (Scrambled) and positive (cMYC) controls were each separately used to transduce OPM2 cells to assess the relative effect of target CDK depletion on cell proliferation or viability. Due to the required throughput for analysis, cytometer to assess for the experimental readout of GFP-positive proportion of this experiment was performed using 96-well plates and a semi-automated flow viable (PI-negative) cells at serial time points. Given the perceived rapidity of constitutive lentiviral transduction and the hypothesis that genetic depletion of transcriptional CDKs such as CDK9 would be associated with rapid induction of apoptosis and loss of GFP representation, the initial experiment was planned with serial readouts at days two, three and four post-transduction. However, it was observed that the proportion of GFP-positive cells was uniformly continuing to increase across all wells at day four post addition of viral supernatant to target cells (Figure 4.17).

Therefore, the CDK RNAi screen was repeated with periodic analysis extending out to 21 days. As it was observed that day four was associated with peak GFP expression

GFP expression at each subsequent time point normalised to day four expression. across all transduced wells, the final analytical readout for this experiment was Some loss of GFP-representation was observed for hairpins targeting cell cycle regulators, CDKs 1, 2, 4 and 6 (Figure 4.18a,b), the transcriptional CDK7 (Figure 4.18b), and the cMYC positive controls (Figure 4.18c). However, the most striking reductions in GFP-representation were from hairpins targeting CDKs 11 and 13 (Figure 4.18c hairpins targeting CDK9. In order to assess whether this lack of phenotype was a ). Unexpectedly, no significant effect was noted from any of the lack of effect of CDK9 depletion or due to non-degradation of the target, validation experiments were next performed.

Transduction of OPM2 cells was repeated and cells harvested at day four for analysis of degradation of target protein (CDK9, CDK11, cMYC) or qRT-PCR where

160 Chapter 4 no commercial antibody was available (CDK13). None of the CDK9-targeting hairpins were associated with depletion of CDK9 protein as assessed by Western blot (Figure 4.19a). Furthermore, only the cMYC hairpin effecting the greatest loss of GFP representation was associated with any appreciable reduction in cMYC protein expression (Figure 4.19a observed with those CDK11 hairpins that were associated with the most marked ). However, a significant reduction of CDK11 was loss of GFP expression (Figure 4.19a). Finally, a reduction of CDK13 mRNA was observed upon qRT-PCR interrogation of those hairpins with the greatest loss of GFP representation (Figure 4.19b).

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Figure 4.17: Preliminary boutique RNAi lentiviral CDK screen using OPM2 cells

Lentivirus targeting CDK1 – CDK13 and non-silencing controls (Scrambled and GAPDH) were applied to transduce exponentially growing OPM2 cells. Flow cytometry was performed at 48, 72 and 96 hour timepoints following addition of viral supernatant for GFP-representation in viable (PI-negative) cells. (a) Representative gating strategy showing cells gated according to single cells (left), PI-negative (middle) and GFP-positive (right). (b) Data from the same experiment shown as bar graphs depicting 48, 72 and 96 hour GFP-positive proportion of viable cells normalized to 48 hour value.

Data representative of mean +/- standard error of the mean for duplicate wells in a single experiment.

162 Chapter 4

a FSC-H Cell No. FSC-A PI GFP b OPM2 3

2

1

0 6 8 8 1 6 4 1 9 3 5 0 2 7 1 0 2 4 7 5 5 1 9 0 0 5 1 2 8 d GAPD H Scramble CDK4_8882 CDK4_8882 CDK1_63668 CDK1_63669 CDK1_64500 CDK1_32859 CDK2_63757 CDK2_63756 CDK2_63757 CDK2_63757 CDK2_64517 CDK2_33664 CDK2_15047 CDK3_63440 CDK3_63440 CDK3_63440 CDK3_63440 CDK3_63440 CDK3_63440 CDK3_30706 CDK4_64169 CDK4_64168 CDK4_64169 CDK4_64595 CDK4_64169 CDK4_64595 CDK4_37478 CDK5_39093 e v i t i s

o 3 P - P

F 2 on G i

t 1 opo r

r 0 8 9 7 2 6 5 0 4 6 5 5 2 3 6 1 0 4 3 6 7 2 9 1 1 4 6 7 5 d P GAPD H Scramble CDK5_6218 CDK5_39093 CDK5_39093 CDK5_39094 CDK6_64503 CDK6_64503 CDK6_63683 CDK6_63682 CDK6_63682 CDK6_63682 CDK6_40408 CDK6_40408 CDK6_40408 CDK6_11290 CDK7_36336 CDK7_36336 CDK7_15048 CDK7_15048 CDK7_15048 CDK7_15048 CDK8_38708 CDK8_38707 CDK8_38708 CDK8_11291 CDK9_63999 CDK9_63999 CDK9_35764 CDK9_35764

3

2

1

0 5 2 5 3 9 9 4 0 1 1 4 6 1 6 5 7 7 9 8 7 2 0 1 2 8 0 7 9 d GAPD H C_36562 C_36561 C_36562 C_36561 C_36561 Scramble CDK13_2438 CDK13_2439 CDK13_2439 CDK13_2439 cM Y cM Y cM Y cM Y cM Y CDK10_64135 CDK10_64135 CDK10_64588 CDK10_37195 CDK10_41019 CDK10_32589 CDK10_37195 CDK10_19419 CDK11_34435 CDK11_33013 CDK11_30682 CDK11_37246 CDK11_31400 CDK12_63908 CDK12_63908 CDK12_63908 CDK12_64539 CDK12_34947 CDK13_30756

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Figure 4.18: Boutique RNAi lentiviral CDK screen using OPM2 cells identifies novel oncogenic dependencies of MYC-driven lymphoid neoplasia

(a-c) Lentivirus targeting CDK1 – CDK13 and non-silencing controls (Scrambled and GAPDH) were applied to transduce exponentially growing OPM2 cells. Flow cytometry was performed at four, seven, 14 and 21 days following addition of viral supernatant for GFP-representation in viable (PI-negative) cells. Data shown as bar graphs depicting four, seven, 14 and 21 day GFP-positive proportion of viable cells normalized to four day value.

Data representative of mean +/- standard error of the mean for duplicate wells from day four and day 21 values for representative construct (2-way ANOVA). one of two independent experiments. *Denotes significant difference comparing

164 Chapter 4

Figure 4.18a Proportion GFP-Positive 0. 0 0. 5 1. 0 1. 5

Scrambled

GAPDH * CDK1_636688

CDK1_636691 *

CDK1_645006 *

CDK1_328594 * CDK2_637571 CDK2_637569 CDK2_637573 CDK2_637575 CDK2_645170

CDK2_336642 * CDK2_150477 CDK3_634401 O PM 2 CDK3_634400 * CDK3_634402 CDK3_634404 CDK3_634407 CDK3_634405 CDK3_307065 CDK4_641691 CDK4_641689 CDK4_641690 CDK4_645950 CDK4_641695 CDK4_645951 CDK4_374782 CDK4_88826

CDK4_88828 *

CDK5_390938 *

165 Chapter 4

Figure 4.18b Proportion GFP-Positive 0. 0 0. 5 1. 0 1. 5

Scrambled GAPDH CDK5_390939 CDK5_390937 CDK5_390942 CDK5_62188 CDK6_645036 CDK6_645035

CDK6_636830 *

CDK6_636824 *

CDK6_636826 *

CDK6_636825 *

CDK6_404085 * CDK6_404082 O CDK6_404083 PM 2 * CDK6_112906 CDK7_363361 CDK7_363360

CDK7_150484 * CDK7_150483 CDK7_150486

CDK7_150487 * CDK8_387082 CDK8_387079 CDK8_387081 CDK8_112911 CDK9_639994 CDK9_639996 CDK9_357647 CDK9_357645

166 Chapter 4

Figure 4.18c Proportion GFP-Positive 0. 0 0. 5 1. 0 1. 5

Scrambled GAPDH CDK10_641355 CDK10_641352 CDK10_645885 CDK10_371953 CDK10_410199 CDK10_325899 CDK10_371954 CDK10_194190 CDK11_344351

CDK11_330131 *

CDK11_306824 *

CDK11_372466 * O PM 2 CDK11_314001 *

CDK12_639086 *

CDK12_639085 * CDK12_639087 CDK12_645397 CDK12_349479

CDK13_24387 *

CDK13_307568 *

CDK13_24392 *

CDK13_24390 *

CDK13_24391 *

cMYC_365622 *

cMYC_365618 * cMYC_365620

cMYC_365617 *

cMYC_365619 *

167 Chapter 4

Figure 4.19: Validation of positive and negative results from boutique RNAi lentiviral CDK screen

Lentivirus targeting CDK9, CDK11, CDK13, cMYC and non-silencing Scrambled control were applied to transduce exponentially growing OPM2 cells. Four days later, non-sorted cells were pelleted from which protein and RNA were extracted from whole cell lysates. (a) separated on gradient polyacrylamide gel using SDS-PAGE prior to immunoblotting Protein extracted from whole cell lysates (10μg) was (b) Quantitative RT-PCR was performed for samples transduced with non-silencing Scrambled control or for CDK11, cMYC, CDK9 and β-Actin loading control. lentivirus targeting CDK13, using two independent primer sets for CDK13 and normalized to the L32 housekeeping gene.

Data obtained from a single experiment.

168 Chapter 4

a 1.330131 1.306824 1.372466 Scrambled CDK9.639994 CDK9.639996 CDK9.357647 CDK9.357645 CDK 1 CDK 1 CDK 1 cMYC.365622 cMYC.365618 cMYC.365620 cMYC.365617 cMYC.365619

CDK11 110 kDa

cMYC 70 kDa

CDK9 42 kDa

-Actin 45 kDa

b CDK13 CDK13 )

d Primer 1 Primer 2 bl e m

a 1.0 r (S c

eve l 0.5 mRNA l

ve 0.0 7 2 7 2 i d d t a l e R 30756 8 30756 8 . . Scramble Scramble CDK13.2438 CDK13.2439 CDK13.2438 CDK13.2439 CDK13 CDK13

169 Chapter 4

4.3 Discussion

4.3.1 CDK9 inhibitors as potent inhibitors of Pol II activation

In vitro and in vivo biomarker studies in this chapter have recapitulated the inhibition of Pol II activation seen with dinaciclib in experiments detailed in associated transcriptional repression of Mcl-1 mRNA. The hypothesis of a linear chapter three. These findings again show the activity of CDK9 inhibitors and druggable pathway involving CDK9 / Pol II regulating Mcl-1 is further implicated as a critical oncogenic requirement for MYC-driven lymphoma.

4.3.2 CDK9 inhibitors potently induce apoptosis of MYC-driven B-cell lymphoma

As was seen with dinaciclib, CDK9 inhibition was observed to potently induce cell autonomous p53-independent apoptosis through in vitro apoptosis assays and also through in vivo biomarker studies. Apoptosis induction was associated with concomitant reduction in Mcl-1, but not Bcl-2 or Bcl-xL protein expression at short time points, further implicating Mcl-1 as the critical oncogenic target of abrogated upon forced retroviral overexpression of various anti-apoptotic proteins, CDK9 inhibition-mediated apoptosis. These apoptotic effects were significantly but not by absence of select BH3-only proteins that were assessed such as Bim or Bmf. If time permitted, repeating the apoptosis assays using Eµ-Myc lymphoma cell lines transduced for overexpression and knockout of each individual apoptosis

CDK9 inhibition are contributory to observed apoptosis induction. Furthermore, family member would have been useful in order to confirm that no other effects of it would have been useful to repeat the apoptosis assays in the presence of caspase protection conferred through inhibition of the downstream apoptosis effector inhibition with the chemical compound QVD-OPh in order to confirm cell death cascade.

The experiments using inducible shCdk9 in Eµ-Myc lymphoma further support the hypothesis of CDK9 being the critical effector of inhibitor-mediated apoptosis. The genetic depletion studies phenocopied the effect of inhibitor with regards to repression of Pol II activation and the detrimental effect on target cells as shown by the competitive culture assays. However, studies are ongoing to show the mechanistic link with reduced Mcl-1 protein expression in this inducible system, and also for a second valid hairpin to confirm accuracy of observations. Given that after exposure to doxycycline ‘switching on’ the hairpin to deplete Cdk9, repeating the cells lose fluorescent protein expression (presumably due to cell death) soon

170 Chapter 4 these genetic experiments in apoptosis-protected cells (previously transduced for overexpression of Bcl-2 or Bcl-xL) is anticipated to allow for assessment of Mcl-1 levels and cytostatic effects following induction of hairpin expression.

4.3.3 CDK9 inhibitors confer differing effects upon cell cycle progression

The cell cycle analyses presented in this chapter provide an interesting insight into the contribution that other targets of the CDK9 inhibitors may be conferring to cell cycle regulation and apoptosis. With regards to the sub-G1 proportion of cells, AZ- CDK9 was more comparable to dinaciclib (Figures 3.2, 4.6b) whereas A1592668.1 was more comparable to palbociclib (Figures 4.4, 4.6a). The AZ-CDK9 effects are likely explained by the reported similarities that this compound possesses to dinaciclib. As with dinaciclib, AZ-CDK9 exhibits potency against CDK9, but also has nanomolar activity against CDKs 1 and 2 as well as GSK3 (Table 1.1) (Cidado et al. 2016).

Protection from apoptosis conferred by A1592668.1 and palbociclib (relative to dinaciclib and AZ-CDK9) may be explained by the CDK4-inhibitory properties of A1592668.1 and palbociclib, whereby CDK4-inhibition may mediate cytostasis conferring relative protection against apoptotic transcriptional effects of CDK9 inhibition. However, this hypothesis is countered by the fact that GSK3-inhibition by dinaciclib/AZ-CDK9 should be removing GSK3-mediated repression of p27Kip1 activity, restoring repression of cyclin D/CDK4 and cyclin E/CDK2 activity leading to cytostasis (Figure 4.20). This would potentiate the cytostasis conferred by dinaciclib/AZ-CDK9 inhibition of CDKs 1 and 2, as well as any potential contribution from CDK9 inhibitor-mediated repression of Pol II transcription of cyclins. Performing the same assays with putative GSK3 inhibitors would have been useful in order to further investigate any contribution of GSK3 inhibition to cytostasis.

4.3.4 CDK9 inhibitors confer differing effects upon Myc transcription

Another striking difference observed between the inhibitors was the effect on cMYC mRNA, whereby dinaciclib and A1592668.1 were associated with reduced cMYC expression, whereas AZ-CDK9 treatment was observed to increase cMYC levels. Explanations for this difference include the possibility that A1592668.1 also possesses bromodomain inhibitory properties akin to dinaciclib and JQ1, or that other off-target effects of AZ-CDK9 are counteracting the early effects that CDK9 inhibition would otherwise have on cMYC mRNA levels.

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GSK3 Dinaciclib / AZ-CDK9

p27Kip1

A1592668.1 / Palbociclib

Cyclin D / CDK4 Cyclin E / CDK2

Cyclin A / CDK1

Cell cycle progression

G1 S

M G2

Figure 4.20: Model of proposed non-CDK9-mediated cytostatic activity of pharmacologic inhibitors of CDK9

GSK3 destabilises p27Kip1, a repressor of cyclin D/CDK4 and cyclin E/CDK2. Black bars denote physiological repression. Red bars denote pharmacologic inhibition. G1, S, G2, M cell cycle phases.

172 Chapter 4

Published studies of PC585 (AZ-CDK9) in human leukaemia cell lines correlate with our observations of increasing cMYC mRNA levels, though suggest a dose- dependent effect on cMYC expression, whereby the lower dose of 100nM was observed to increase cMYC mRNA at six hours, whereas higher doses were associated with potent repression of cMYC (Garcia-Cuellar et al. 2014). Remarkably, when inhibitory properties), the same effect of increased cMYC at lower concentration the same group assessed flavopiridol (which does not possess bromodomain- and reduction at higher concentration was observed. Furthermore, longer periods of CDK9 inhibition of HeLa cells with a different inhibitor, i-CDK9, have been shown to lead to PTEFb-dependent upregulation of cMYC contingent upon BRD4 activity (Lu et al. 2015).

have dynamic effects on cMYC expression in a dose-dependent, and perhaps time- Taken together with our findings, these results suggest that CDK9 inhibition may dependent manner. Transcriptional regulation of MYC activity (including the role advanced technologies such as global run-on sequencing in order to assess the of CDK9) is a significant focus of ongoing research in the laboratory as we pursue dynamic effects on nascent RNA that occur upon inhibition of transcriptional machinery. These ongoing studies include time course experiments and assessment of variable doses of the various CDK9 inhibitors with multiple readouts including the effects on cMYC and MCL1 expression.

4.3.5 CDK9 inhibition represents a tolerable and effective therapy against MYC-driven B-cell lymphoma in vivo

It would be predicted that inhibition of global transcriptional processes dependent

This hypothesis is supported by the fact that the Pol II inhibitor, α-amanitin, is on Pol II would be associated with significant toxicities to non-malignant tissues. the chemical within Amanita phalloides (death cap mushrooms) that confers potentially fatal hepatotoxicity (Wieland 1968; Brueckner and Cramer 2008).

However, the in vivo studies of CDK9 inhibitors in this chapter not only highlighted the targeted activity of these agents with a predominant effect on short-lived proteins, but also demonstrated tolerability and efficacy in an orally bioavailable formulation. These findings are further supported by independent groups in MLL-rearranged leukaemia (Garcia-Cuellar et al. 2014) and MYC-driven contemporaneously publishing on the efficacy and tolerability of CDK9 inhibitors hepatocellular carcinoma (Huang et al. 2014). Hence, despite targeting global transcriptional machinery, a therapeutic window exists in which these inhibitors may have preferential efficacy against tissues that possess a greater dependency 173 Chapter 4 upon these short-lived proteins.

4.3.6 Transcriptional kinases represent bona fide therapeutic targets in MYC-driven lymphoid malignancies

Consistent with the pharmacologic inhibitor experiments, the genetic studies performed in this chapter also support the hypothesis of targeting transcriptional kinases in MYC-driven lymphoid malignancy. The inducible CDK9 silencing hairpins in Eµ-Myc lymphoma demonstrated the detrimental effect that CDK9 depletion has upon this system, while the lack of phenotypic change in the RNAi lentiviral experiment is readily explained by the validation experiment where it was shown that the algorithm-generated hairpins targeting CDK9 did not in fact reduce CDK9 to any significant extent. CDK11 and CDK13 as critical oncogenic requirements of MYC-driven lymphoid A striking observation from the boutique RNAi screen was the identification of malignancy. While little literature exists pertaining to the function and regulation of these CDKs, both are involved with regulation of Pol II activity. CDK11 / cyclin L has recently been shown to control interaction of the S-mediator and kinase modules to form the L-mediator complex which is a co-activator of Pol II (Drogat et al. 2012). Furthermore, CDK11 activity has been demonstrated to be a critical requirement of models of breast cancer, osteosarcoma and multiple myeloma (Tiedemann et al. 2012; Duan et al. 2012; Feng et al. 2014; Zhou et al. 2014; Chi et al. 2015). CDK13 is a Pol II CTD kinase that possesses promiscuous activity through phosphorylation of both serine 2 and serine 5 in cells primed with pre- phosphorylation of serine 7 (Greifenberg et al. 2016). An oncogenic role has hepatocellular carcinoma (Kim et al. 2012). Despite these recent associations of been implicated through identification of frequent amplification of CDK13 in CDK11 and CDK13 postulating oncogenic roles, no directed therapies have yet been developed and this represents a promising novel therapeutic approach for these diseases, and perhaps more broadly to transcriptionally-addicted malignancies.

4.3.7 Bcl-2 antagonism accelerates MYC-driven lymphoma progression

Much excitement has followed the initial results of BCL-2 antagonism with venetoclax in the treatment of chronic lymphocytic leukaemia (Roberts et al. 2016). BCL-2 is often overexpressed in DLBCL (Johnson et al. 2012b), and the combination of BCL2 and cMYC translocations herald the extremely poor prognosis of ‘double-hit’ lymphoma (Oki et al. 2014). Hence, CDK9 inhibitor- mediated repression of MCL-1 and pharmacologic antagonism of BCL-2 would

174 Chapter 4 seem a rational therapeutic strategy. Indeed, this combination was observed to be synergistic when assessed by an independent research group using an engineered lymphoma model of MYC/BCL2 overexpression (Li et al. 2015). However, due to the engineered dependency of that model on BCL2, sensitisation to BCL2 inhibition would be predicted and indeed single agent activity was also observed in their studies. While the combination in our studies was observed to be tolerable and effective, the single arm control of Bcl-2 inhibition with ABT-199 was found to accelerate disease progression and reduce overall survival of mice in two Myc-driven lymphoid models. This is of concern, as the success of venetoclax has led to early phase clinical trials combining it with standard chemotherapeutic backbones (without CDK9 inhibition) for the treatment of DLBCL, and furthermore demonstrated Richter’s transformation to aggressive lymphoma while on therapy a small though significant proportion of patients that received venetoclax for CLL

(Roberts et al. 2016). Our findings suggest that the empiric addition of this novel compensatory pathways leading to disease acceleration. We await the results of targeted therapy without previous demonstration of efficacy may be upregulating these early phase clinical trials with interest.

The mechanism of disease acceleration conferred by Bcl-2 inhibition was not pursued further during experimental work for the preparation of this thesis. Several potential mechanisms may be contributory, as Bcl-2 has also been shown to have non-canonical roles regulating cell cycle progression and tumour suppression (Huang et al. 1997; La Coste et al. 1999; Vairo et al. 2000). Overexpression of regulators, p27 and p130, leading to reduced progression through the G1/S Bcl-2 in fibroblasts is associated with an increase in expression of the (Vairo et al. 2000). This cytostatic effect is conferred through the BH4 domain and is distinct from the anti-apoptotic function of Bcl-2 (Huang et al. 1997). Direct human relevance of this cytostatic function has been demonstrated to silence the cytostatic effects of translocated BCL-2 when follicular lymphoma with the finding that somatic mutation of this region is a relatively frequent event undergoes Richter’s transformation to aggressive lymphoma (Tanaka et al. 1992). It is yet to be established whether pharmacologic inhibition of BCL-2 with ABT- 199 also interferes with the BH4 cytostatic properties of this protein.

in vivo experiments of ABT-199 with or without CDK9 inhibition for the purposes of in vivo assays to assess the relative It would have been beneficial to repeat the effect that Bcl-2 antagonism is having upon expression of other anti-apoptotic family members. Aside from the non-canonical properties of Bcl-2 described above, another simple explanation for observed disease acceleration is that Bcl-

175 Chapter 4

2 antagonism may be further stimulating these lymphomas to upregulate Mcl- 1, which is already a critical oncogenic dependency. Should this indeed be the mechanism, it would be interesting to assess whether pre-treatment of MYC- driven B-cell lymphoma with a Bcl-2 inhibitor would further reprogram the cells for greater Mcl-1 dependency, conferring increased sensitivity to indirect or direct Mcl-1 antagonism. However, unpublished data recently presented by Professor David Huang (D. Huang 2016, personal communication, 13 April) of the Walter and Eliza Hall Institute suggests that myeloma cell lines exposed to incrementally increased doses of ABT-199 in vitro Bax expression as a mechanism of resistance, and this or reduced Bak may also are demonstrating a significant reduction in account for the disease acceleration observed in our in vivo experiments.

4.3.8 Conclusion

Experimental work depicted in this chapter has further supported the hypothesis of CDK9 as a druggable oncogenic requirement of MYC-driven lymphoma. These experiments have complemented those of chapter three in showing that selective CDK9 inhibition replicates the in vitro and in vivo effects of dinaciclib, further reinforcing CDK9, and not off-target activity, to be the critical mechanism of dinaciclib-induced apoptosis. Furthermore, the implication of CDK11 and CDK13 as other oncogenic requirements of MYC-driven lymphoma suggests a state of transcriptional addiction that may be disrupted through either inhibition of kinase sites directly or through disruption of the Pol II elongation complex. These of transcriptional machinery separately or synergistically as a novel therapeutic findings provide a rationale for further studies targeting different components approach to MYC-driven disease.

Finally, the critical oncogenic dependency of this subset of disease upon anti- apoptotic MCL-1 also provides rationale for development of direct MCL-1 inhibitors, to directly target. In the next chapter, MCL-1 is pharmacologically and genetically which, in contrast to other BCL-2 family members, has previously proven difficult bona fide therapeutic option for treatment of MYC-driven lymphoma. targeted to confirm its antagonism as a

176 Chapter 5: Genetic and pharmacologic targeting of MCL-1 in MYC-driven models of B-cell lymphoma

177 Chapter 5

5.1 Introduction

The previous two chapters have demonstrated activity of a number of pharmacologic inhibitors of CDK9 in MYC-driven models of B-cell lymphoma. The associated reduction in MCL-1 expression is postulated to be the critical mechanism of activity, though other downstream effectors are not excluded as being contributory. MCL-1 itself has proven to be an elusive direct drug target, in vitro testing of a putative MCL-1 inhibitor occurred towards the latter period of time in which this thesis and the first publications on the development and was produced (Leverson et al. 2014; Xiao et al. 2015). In the similar period of

MYC-driven lymphoma has upon MCL-1 expression (Kelly et al. 2014; Aubrey et time, extensive genetic studies have confirmed the oncogenic dependency that al. 2015; Grabow et al. 2016a; 2016b). However, no reports of in vivo activity and tolerability of MCL-1 inhibitors have yet been published.

II-independent antagonism of MCL-1. Firstly, direct MCL-1 antagonism without Experiments performed in this chapter assess the efficacy and tolerability of Pol perturbation of global transcriptional processes (Figure 5.1) is demonstrated

MYC-driven models of lymphoma. Secondly and despite the extensive literature as sufficient to potently induce p53-independent, cell autonomous apoptosis in outlining the dependency of a broad array of tissues upon MCL-1 activity, in vivo studies described herein demonstrate the presence of a therapeutic window when targeting MCL-1, and the significant anti-lymphoma activity that a putative small MCL-1 to be a bona fide therapeutic target in MYC-driven B-cell lymphoma, and molecule BH3-mimetic targeting MCL-1 possesses. These observations confirm support the rationale for clinical development of MCL-1 inhibitors in oncology.

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PTEFb Cyclin T1 CDK9 MYC YSPTSPS/YSPTSPS RNA polymerase II

Transcription

MCL-1

MCL-1-inhibitors

Figure 5.1: Targeting MCL-1 in a transcriptionally-addicted model of MYC-driven lymphoma

Schematic representation of the proposed oncogenic pathway demonstrating MYC binding of cyclin T1 to recruit P-TEFb to MYC transcriptional target sites. MCL-1 is represented as a critical oncogenic dependency of this model. MCL-1 inhibition (red bar) and genetic interference of MCL-1 (red cross) are hypothesized to potently induce apoptosis. P denotes phosphorylation.

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5.2 Results

5.2.1 MCL-1 inhibition potently induces apoptosis of Eµ-Myc lymphoma and human IG-cMYC-translocated lymphoma in vitro

Having shown CDK9-mediated repression of MCL-1 to be associated with potent induction of apoptosis in previous chapters, it would be expected that direct inhibition of MCL-1 with a selective inhibitor would also potently induce intrinsic apoptosis. In order to interrogate this further, a selective MCL-1 inhibitor (AZ-

MCL1) and BCL-2 / BCL-XL inhibitor (AZD4320) were sourced from Astra Zeneca Table 5.1 (Waltham, MA, USA). The inhibitory profiles of these inhibitors are shown in them to be selective small molecule inhibitors (Belmonte et al. 2014). and initial preclinical studies from our industry collaborators have confirmed

In vitro apoptosis assays revealed nanomolar concentrations of AZ-MCL1 to induce apoptosis of Eµ-Myc lymphoma cell lines in a p53-independent manner (Figure 5.2a). However, overexpression of Mus musculus Mcl-1 or Bcl-2 driven by a retroviral promoter (Figures 3.5b, 5.2b inhibitor-mediated apoptosis (Figure 5.2a). In contrast, Eµ-Myc lymphoma was ) was sufficient to significantly abrogate insensitive to inhibition with the BCL-2 / BCL-XL inhibitor AZD4320, indicating that these proteins are not critical to survival of these lymphomas (Figure 5.2c). Only upon forced overexpression of Bcl-2 were these lymphomas reprogrammed for sensitivity to Bcl-2 inhibition with AZD4320 (Figure 5.2c are consistent with previous published pharmacologic and genetic studies ). These findings interrogating the effect of Bcl-2 / Bcl-XL inhibition or deletion in Eµ-Myc lymphoma (Mason et al. 2008; Kelly et al. 2014; Whitecross et al. 2009), and reinforce the selectivity of the respective inhibitors.

In order to assess whether inter-species divergence of Mcl-1 could be responsible for the protection observed above, an extensive suite of derived Eµ-Myc lymphomas overexpressing different Homo sapiens anti-apoptotic proteins was assessed for apoptosis induction upon in vitro exposure to AZ-MCL1. Overexpression of Homo sapiens MCL-1 was observed to sensitise Eµ-Myc lymphoma to MCL-1 inhibition even relative to the parental wild-type cell line (Figure 5.3a,b). As anticipated, overexpression of anti-apoptotic BCL-2, BCL-XL, A1 and BCL-W were all protective against AZ-MCL1-mediated apoptosis (Figure 5.3a,b).

It is expected that observed cell death upon AZ-MCL1 inhibition is effected through intrinsic apoptosis induction. In order to further support this, nuclear

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IC50 [nM]

AZ-MCL1 AZD4320

MCL-1 <3 >1000

BCL-2 28000 <10

BCL-XL >33000 <10

Table 5.1: Inhibitory profile of the MCL-1 inhibitor AZ-MCL1

In vitro proteins MCL-1, BCL-2 and BCL-X (personal communication Matthew Belmonte, inhibitory profiles of AZ-MCL1L and AZD4320 for key anti-apoptotic

AstraZeneca, Waltham, MA, USA). Results derived from Bim fluorescence resonant assay (BCL-2, BCL-X ). energy transfer bindingL assay (MCL-1) and Bim fluorescence polarization binding

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Figure 5.2: In vitro selectivity of MCL-1 and BCL-2 / BCL-XL inhibitors

(a) Myc lymphoma cell line #4242 was stably transduced with expression vectors MSCV-Mcl-1-GFP (murine), MSCV-Bcl-2-GFP (murine) or empty vector Eμ- (denoted as # Myc lymphoma cell line #3391 were

4242). Derived cell lines and Eμ- cytometric analysis for annexin-V / PI uptake. (b) -Myc lymphoma cell line incubated with varying concentrations of AZ-MCL1 for 24 hours prior to flow #4242 was stably transduced with expression vectors MSCV-Bcl-2-GFP (murine) or Eμ empty vector (denoted as #4242). Protein extracted from whole cell lysates (10µg) was separated using SDS-PAGE prior to immunoblotting for Bcl-2 and tubulin. (c) -Myc lymphoma cell line #4242 was stably transduced with expression vectors MSCV-Mcl-1-GFP (murine), MSCV-Bcl-2-GFP (murine) or empty vector (denoted as Eμ # Myc lymphoma cell line #3391 were incubated

4242). Derived cell lines and Eμ- analysis for annexin-V / PI uptake. with varying concentrations of AZD4320 for 24 hours prior to flow cytometric

Data representative of mean +/- standard error of the mean of non-viable cells for p<0.05 comparing equal drug concentrations between the representative genotypes and vector control (2-way ANOVA). three independent experiments. *

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a #4242 #4242tmMcl-1 #4242tmBcl-2 #3391 p53-/- 100 I

/ P * n * * * * * * * * * * * * 50 nn ex i A

%

0 2 3 2 3 2 3 2 3 0 0 0 0 3 6 3 6 3 6 3 6 12 5 25 0 50 0 12 5 25 0 50 0 12 5 25 0 50 0 12 5 25 0 50 0 100 0 100 0 100 0 100 0

AZ-MCL1 [nM] b t mBcl-2 4242 4242 # #

Tubulin 52 kDa

Bcl-2 26 kDa

c #4242 #4242tmMcl-1 #4242tmBcl-2 #3391 p53-/- 100

I * * / P n * 50

nn ex i * * * * * * A

%

0 2 3 2 3 2 3 2 3 0 0 0 0 3 6 3 6 3 6 3 6 12 5 25 0 50 0 12 5 25 0 50 0 12 5 25 0 50 0 12 5 25 0 50 0 100 0 100 0 100 0 100 0

AZD4320 [nM]

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Figure 5.3: In vitro selectivity of select MCL-1 inhibitor against a panel of derived Eµ-Myc cell lines

(a) Myc lymphoma cell line #4242 was stably transduced with expression vectors MSCV-hMCL-1-GFP (#4242thMCL-1), MSCV-hBCL-2-GFP (#4242thBCL-2), Eμ- # # MSCV-hBCL-XL-GFP ( 4242thBCL-XL), MSCV-hA1-GFP ( 4242thA1), MSCV-hBCL-W- GFP (#4242thBCL-W) or empty vector (denoted as #4242). Derived cell lines and Myc lymphoma cell line #3391 were incubated with varying concentrations of h, Eμ- Homo sapiens. (b) Representative derived cell lines #4242thMCL-1, #4242thBCL-2, AZ-MCL1 for 24 hours prior to flow cytometric analysis for annexin-V / PI uptake. # # # 4242thBCL-XL and 4242 transduced with empty vector (denoted as 4242) were exponentially cultured prior to protein extraction from whole cell lysates. Protein immunoblotting for human MCL-1, human BCL-2, human BCL-X and tubulin (10μg) was separated on gradient polyacrylamide gel using SDS-PAGEL prior to loading controls.

Data representative of mean +/- standard error of the mean of non-viable cells for p<0.05 comparing equal drug concentrations between the representative genotypes and vector control (2-way ANOVA). three independent experiments. *

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a #4242 #4242t #4242t #4242t #4242t #4242t #3391 -/- hMCL-1 hBCL-2 hBCL-XL hA1 hBCL-W p53 * * 100 * I / P n 50

nn ex i * * * * * * * * * * * * * * * A

%

0 0 0 0 0 0 0 0 12 5 25 0 50 0 12 5 25 0 50 0 12 5 25 0 50 0 12 5 25 0 50 0 12 5 25 0 50 0 12 5 25 0 50 0 12 5 25 0 50 0 100 0 100 0 100 0 100 0 100 0 100 0 100 0 AZ-MCL1 [nM] b t hMCL-1 t hBCL-2 4242 4242 4242 4242 # # # #

MCL-1 40 kDa BCL-2 26 kDa

Tubulin 52 kDa Tubulin 52 kDa

L t hBCL-X 4242 4242 # #

BCL-XL 30 kDa

Tubulin 52 kDa

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DNA fragmentation and mitochondrial outer membrane permeabilisation were assessed as independent readouts for in vitro apoptosis. As expected, exposure of Eµ-Myc lymphoma to increasing concentrations of AZ-MCL1 was seen to increase the proportion of apoptotic cells as shown by nuclear DNA fragmentation (cells in subG1 gate) and loss of mitochondrial membrane potential (Figure 5.4). These intrinsic apoptosis induction. findings support the hypothesis that MCL-1 inhibition kills target cells through

To demonstrate direct relevance to human lymphoma, a suite of human IG- cMYC-translocated Burkitt lymphoma and multiple myeloma cell lines were also assessed for MCL-1 inhibitor mediated apoptosis (Figure 5.5a). Notably, the

LD50 was reduced by almost two doubling dilutions relative to that observed in prior Eµ-Myc studies, indicating marked sensitivity of these human MYC-driven lymphoid malignancies to MCL-1 inhibition. In contrast, BCL-2 / BCL-XL inhibition was ineffective at inducing apoptosis in these Burkitt cell lines (Figure 5.5b).

5.2.2 MCL-1-inhibitor treatment of Eµ-Myc lymphoma is associated with biomarkers of rapid intrinsic apoptosis induction

To further interrogate and validate the mechanism of action of AZ-MCL1, proteins from cell lysates of in vitro treated and untreated Eµ-Myc lymphoma cell lines were immunoblotted to assess for markers of apoptosis activation. Initially, Eµ- Myc cells were cultured with DMSO or AZ-MCL1 for four hours prior to harvesting of cell pellets and preparation for Western blotting as previously described. A marked increase in cleaved PARP was observed in treated cells with a concomitant subtle reduction in Mcl-1 and no change in Bcl-2 protein expression (Figure 5.6a). In order to assess the time to induction of apoptosis with this model, a time course experiment was performed where Eµ-Myc cells were cultured for different lengths of time prior to cell lysate preparation. Strikingly, increased cleaved PARP was observed at as soon as 15 minutes after exposure to AZ-MCL1 (relative to DMSO treatment) and a greater proportion of PARP was cleaved than uncleaved at one- hour incubation or longer (Figure 5.6b). These experiments further demonstrate rapid induction of intrinsic apoptosis upon exposure of Eµ-Myc cells to select Mcl- 1 inhibition.

In contrast to the transcriptional inhibitory properties of CDK9 inhibitors, MCL-1 inhibition would not be predicted to be associated with any effect on MCL-1 mRNA levels. In order to demonstrate this, Eµ-Myc lymphoma cells were exposed to AZ- MCL1 in vitro prior to RNA extraction and assessment for any effect on Mcl-1 or

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Bcl-2 Mcl-1 or Bcl-2 mRNA was observed following three hour incubation of Eµ-Myc lymphoma with the MCL- mRNA. As anticipated, no significant change in either 1 inhibitor (Figure 5.7).

5.2.3 MCL-1-inhibitor therapy is tolerated and induces apoptosis of Eµ- Myc lymphoma in vivo

Our industry collaborator (Ammar Adam, Astra Zeneca, Waltham, MA, USA) had performed extensive testing of AZ-MCL1 in immunocompromised mice and found a dose of 100mg/kg AZ-MCL1 in 30% HPBCD administered weekly for two weeks weight (data not shown). The major reported toxicity from their studies was of by intravenous tail vein injection to be well tolerated with no significant loss of tail necrosis at the injection site if extravasation of the injected compound were to occur. Prior to undertaking therapy studies in tumour-bearing mice, a pilot maximum tolerated dose experiment was performed in non-tumour-bearing C57BL/6 mice to ensure safety of delivery in our in vivo models. Mice received a total of two doses of AZ-MCL1 100mg/kg or vehicle given one week apart. While Figure 5.8a), one episode of extravasation of drug during IV administration did occur and no significant loss of weight or other sign of systemic toxicity was observed ( Figure 5.8b). led to significant necrosis at the tail injection site the following day ( When mice were transplanted with GFP-expressing Eµ-Myc lymphoma and left to establish bulky lymph nodes prior to a single dose of vehicle or AZ-MCL1 (Figure 5.9a), in vivo assays showed loss of GFP-expressing lymphoma cells with AZ-MCL1 of this observation (Figure 5.9b). However, blood sampling also revealed hepatic treatment though an obvious outlier in each group prevented statistical significance enzyme elevation (alanine transaminase, aspartate aminotransferase), pancreatic enzyme elevation (amylase, lipase) and general muscular damage (creatine kinase) associated with AZ-MCL1 as measured by laboratory biochemistry testing, indicative of organ toxicity (Figure 5.9c).

5.2.4 MCL-1 inhibitor therapy is associated with prolonged survival of tumour-bearing mice in vivo

therapy in the clinic, therapy studies were next performed to ascertain whether To demonstrate that the observed efficacy of AZ-MCL1 may translate to an effective AZ-MCL1 may confer a survival advantage to mice bearing Eµ-Myc lymphoma. Our collaborator (Ammar Adam, Astra Zeneca, Waltham, MA, USA) had also provided data on in vivo use of the BCL-2 / BCL-XL inhibitor, AZD4320 (data not shown),

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Figure 5.4: In vitro readouts of apoptosis induction for Eµ-Myc lymphoma upon pharmacologic MCL-1 inhibition

(a) Exponentially growing Eµ-Myc lymphoma #3391 was exposed to varying concentrations of AZ-MCL1 for 24 hours prior to cell preparation using the Nicoletti protocol for analysis of nuclear DNA content as represented by PI of cells with increasing concentration of AZ-MCL1. (b) Cells from the same uptake. Representative flow cytometry plots show increase in subG1 fraction experiment were analysed for mitochondrial membrane potential as assessed cytometry plots show increase in TMRE-negative cell proportion with increasing by tetramethylrhodamine, ethyl ester (TMRE) uptake. Representative flow concentration of AZ-MCL1. (c) Bar graphs depict proportion of cells in subG1 gate (left) and proportion of TMRE-negative cells (right) from experiments described above.

Data representative of mean +/- standard error of the mean of apoptotic cells for three independent experiments.

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a 250 nM 500 nM 1000 nM Cell No.

PI b 250 nM 500 nM 1000 nM Cell No. TMRE c

#3391 #3391 100 * 100 * e v i t a 1 g e

* n

ub G 50 50 E S

R % M T

% 0 0 0 0 12 5 25 0 50 0 12 5 25 0 50 0 100 0 100 0 AZ-MCL1 [nM] AZ-MCL1 [nM]

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a BL-41 Ramos Namalwa OPM2 H929 100 * * * * * * I

/ P * * * * n 50 nn ex i A

%

0 6 2 3 6 2 3 6 2 3 6 2 3 6 2 3 0 0 0 0 0 1 3 6 1 3 6 1 3 6 1 3 6 1 3 6 12 5 12 5 12 5 12 5 12 5

AZ-MCL1 [nM]

b BL-41 Ramos 100 I / P n 50 nn ex i A

%

0 0 0 12 5 25 0 50 0 12 5 25 0 50 0 100 0 100 0 AZD4320 [nM]

Figure 5.5: MCL-1 inhibition, but not BCL-2 / BCL-XL inhibition, is associated with apoptosis induction of human IG-cMYC- translocated lymphoid malignancy in vitro

(a) Human IG-cMYC-translocated Burkitt lymphoma cell lines (grey bars) and multiple myeloma cell lines (white bars) were cultured in exponential growth phase in the presence of varying concentrations of AZ-MCL1 for 24 hours prior (b) Human IG-cMYC- translocated Burkitt lymphoma cell lines were cultured in the presence of varying to flow cytometric analysis for annexin-V / PI uptake. annexin-V / PI uptake. concentrations of AZD4320 for 24 hours prior to flow cytometric analysis for

Data representative of mean +/- standard error of the mean of non-viable cells for p<0.05 comparing drug to vehicle control for each individual cell line (2-way ANOVA). three independent experiments. *

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a DMSO AZ-MCL1

PARP 116 kDa Cleaved PARP 89 kDa

Mcl-1 40 kDa

Bcl-2 26 kDa

Tubulin 52 kDa

b AZ-MCL1 3h DMSO 15m 30m 1h 2h 4h

PARP 116 kDa Cleaved PARP 89 kDa

Tubulin 52 kDa

Figure 5.6: AZ-MCL1 treatment is associated with biomarkers of rapid apoptosis induction

(a) Eµ-Myc #4242 lymphoma cells were incubated for four hours in the presence of AZ-MCL1 500nM or DMSO vehicle prior to Western blotting. Protein extracted

SDS-PAGE prior to immunoblotting for PARP, MCL-1, BCL-2 and tubulin loading from whole cell lysates (10μg) was separated on gradient polyacrylamide gel using control. (b) Eµ-Myc lymphoma #4242 was incubated for various lengths of time in the presence of AZ-MCL1 500nM or DMSO vehicle prior to Western blotting. polyacrylamide gel using SDS-PAGE prior to immunoblotting for PARP and tubulin Protein extracted from whole cell lysates (10μg) was separated on gradient loading control. m, minutes; h, hours.

Data representative of three independent experiments.

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Bcl-2 Mcl-1

) NS NS 1.5 MS O D ( 1.0 eve l

mRNA l 0.5 ve i t a l e

R 0.0 1 1 DMSO DMSO AZ-MCL AZ-MCL

Figure 5.7: Selective MCL-1 inhibition is not associated with transcriptional changes to anti-apoptotic mRNA expression

Eµ-Myc #4242 lymphoma cells were cultured in the presence of AZ-MCL1 500nM or DMSO vehicle for three hours prior to RNA extraction. Quantitative RT-PCR was performed using primer sets for Bcl-2 (left) and Mcl-1 (right) and normalized to the GAPDH

housekeeping gene. NS, not significant (student’s t-test). Data representative of mean +/- standard error of the mean for three independent experiments.

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a b NS

) 150 e

ase li n 100 b ( gh t

i 50 e W

% 0 e 1 Vehicl AZ-MCL

Figure 5.8: AZ-MCL1 therapy in vivo is well tolerated systemically and associated with specific toxicity

(a) Non-tumour bearing C57BL/6 mice were dosed with vehicle 30% HPBCD or AZ- MCL1 100mg/kg, weekly by intravenous tail vein injection for two weeks. Weights of individual mice at completion of two weeks of therapy relative to baseline (b) Photograph depicting local necrosis at the injection site due to extravasation of AZ-MCL1 at the time of administration. weights are shown. NS, not significant.

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Figure 5.9: AZ-MCL1 therapy in vivo is associated with selective apoptosis of lymphoma cells and biochemical evidence of organ toxicities

(a) Non-irradiated C57BL/6 recipient mice were transplanted by tail vein injection with 1 x 105 Myc #4242 lymphoma cells harvested from lymph nodes of a syngeneic mouse. After allowing 13 days to establish bulky lymph nodes, GFP-expressing Eμ- mice received a single dose of vehicle 30% HPBCD (n=5) or AZ-MCL1 100mg/kg (n=5), three hours prior to being bled to examine for biochemical toxicities of (b) Lymph nodes were harvested from independent mice and prepared as a single cell suspension from which non-malignant cell therapy and being sacrificed. and lymphoma cell viability were examined according to GFP expression of cells (denoting viability of lymphoma cells) in the predicted gates according to morphology. Flow cytometry plots show lymphoma cells in the low side scatter (SSC) and high forward scatter (FSC) gates (left), and the proportion GFP-positive within that gate (right). Upper panels are from a representative vehicle-treated mouse, and lower panels from a representative MCL-1-treated mouse. Scatter plot (far right) shows the GFP-positive proportion of cells in the lymphoblast gate, comparing vehicle to AZ-MCL1-treated cohorts. (c) Biochemistry assessment from serum of mouse liver function tests (aspartate aminotransferase, AST; and alanine transaminase, ALT), pancreatic (amylase and lipase) and muscle U/L units per litre. fiber injury (creatine kinase, CK).

p p<0.0001 (student’s t-test). Data representative of mean +/- standard error of the mean. NS, not significant; *** <0.001, ****

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a Eµ-Myc 1x105 cells

Day 13 +3h

30% HPBCD vehicle AZ-MCL1 100mg/kg b

e 100 NS v i t i po s

50 P F G

% 0 e 1 Vehicl SSC Cell No. FSC GFP AZ-MCL c 3000 400 NS 1500 NS *** /L ) U ( /L ) /L ) 300 2000 1000 U U ( ( 200 T ase l LT S 1000 500

A 100 A

0 0 Am y 0 e e 1 e 1 1 Vehicl Vehicl Vehicl AZ-MCL AZ-MCL AZ-MCL

200 **** 2000 *** /L )

U 150 1500 /L ) ( U 100 ( 1000

50 CK 500 Lip ase 0 0 e e 1 1 Vehicl Vehicl AZ-MCL AZ-MCL

195 Chapter 5 and this was used as a control therapy at the tolerated and recommended dose of 10mg/kg weekly by tail vein injection in 30% HPBCD vehicle.

Syngeneic mice were transplanted with Eµ-Myc lymphoma #4242 and treatment commenced three days later as per previous therapy experiments (Figures 3.7, 4.11). A total of two doses of AZ-MCL1, AZD4320 or vehicle were administered on day three and day 10 post-transplantation, and biomarkers of disease of disease progression as measured by GFP-representation and absolute number progression sought. AZ-MCL1 therapy was associated with significant abrogation of lymphoma cells in leukaemic phase at day 11 post-transplantation (Figure 5.10a,b

). Furthermore, despite a small statistically, but not clinically, significant in AZ-MCL1 treated mice, there was a relative preservation of platelet count reduction in haemoglobin and significant reduction in neutrophil count observed (Figure 5.10c). Of note, AZD4320-treated mice demonstrated no reduction in disease activity relative to vehicle-treated control mice (Figure 5.10a,b), though with vehicle-treated control mice (Figure 5.10c). Following the two doses of displayed a statistically significant reduction in platelet count when compared therapy or vehicle, mice were observed until development of signs of progressive advantage was conferred by BCL-2 / BCL-X lymphoma requiring sacrifice as previouslyL described. While no overall survival survival advantage was seen for mice treated with the MCL-1 inhibitor (Figure inhibition with AZD4320, a significant 5.10d).

Given the short half-life of MCL-1, the question of therapy scheduling was next previous experiment versus a schedule of two sequential days of dosing (Figure addressed to compare efficacy of therapy administered one week apart as per the 5.11a). The #3391 Eµ-Myc lymphoma clone was used in order to also show MCL- 1 inhibition to be effective in a p53-null model. Both schedules of AZ-MCL1 were

p53-independent associated with a significant prolongation of overall survival relative to vehicle- activity (Figure 5.11b). Remarkably, the dose-dense schedule of doses treated controls, confirming MCL-1 inhibitor therapy to have administered on only days three and four post-transplantation was observed to cure 50% of treated mice.

Finally, in order to show relevance of this approach to human lymphoma in vivo, a therapy experiment was performed using a bioluminescence-tagged human IG-cMYC-translocated Burkitt lymphoma cell line xenografted into immunocompromised mice. Bioluminescence imaging performed at day 14 post- transplantation and following 11 days treatment with AZ-MCL1 showed abrogation

196 Chapter 5 of disease progression relative to mice treated with HPBCD vehicle control (Figure 5.12a overall survival advantage was conferred when compared to mice treated with ). Following two doses of AZ-MCL1 inhibitor therapy, a significant vehicle (Figure 5.12b). This experiment demonstrates in vivo relevance of MCL-1 inhibitor therapy in a model of human MYC-driven B-cell lymphoma.

5.2.5 Genetic depletion of Mcl-1 is detrimental to MYC-driven lymphoma

Having shown efficacy of pharmacological MCL-1 inhibition against MYC-driven a critical oncogenic requirement of MYC-driven lymphoma through genetic de- lymphoma models, the final experiments were performed to confirm MCL-1 as induction. Recently published data utilising an inducible CRISPR/Cas9 platform to target MCL1 has shown a reduction in viability of two target Burkitt lymphoma cell lines upon depletion of MCL-1 (Aubrey et al. 2015). As Eµ-Myc lymphoma was the predominant model utilised in earlier experiments, this model was used for the genetic de-induction studies described below.

Previously validated and published silencing short hairpin RNAs targeting Mcl1 (Fellmann et al. 2013) were sourced from a collaborator (Associate Professor Ross Dickins, Australian Centre for Blood Diseases, Monash University, Victoria, Australia) and cloned into the REBIR construct previously described in chapter four (section 4.2.5). The resultant inducible shRNAs targeting MCL-1 were then transduced into Eµ-Myc lymphoma cells. Transduced cells were placed in a competitive culture assay with non-transduced cells, and following tetracycline induction of the silencing hairpin for just 24 hours, a rapid loss of dsRED reduction was observed in the non-silencing control (Figure 5.13 representation was observed for the hairpin targeting Mcl-1, whereas no significant Myc lymphoma. ). These findings further confirm Mcl-1 as a critical oncogenic requirement of Eµ-

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Figure 5.10: MCL-1 inhibitor therapy is associated with reduced disease progression and prolongation of survival for mice transplanted with Eµ-Myc lymphoma

Non-irradiated C57BL/6 recipient mice were transplanted by tail vein injection with 1 x 105 Myc #4242 lymphoma cells harvested from lymph nodes of a syngeneic mouse. Therapy was commenced three days later with 30% HPBCD Eμ- vehicle, AZD4320 10mg/kg or AZ-MCL1 100mg/kg, administered weekly by intravenous tail vein injection for two weeks. (a) Retro-orbital blood sampling at day 11 post-transplantation was analysed for GFP-representation of lymphoblasts in the peripheral blood (gated according to morphology of viable cells in the low side-scatter and mid-forward scatter region). (b) Full blood examination at day 11 indicating the absolute number of lymphocytes (lymphoblasts in leukaemic phase) in the peripheral blood. (c) Full blood examination at day 11 indicating effect of each therapy on haemoglobin (upper left), neutrophil count (right) and platelet count (lower left). (d) Kaplan-Meier curve showing overall survival of mice in each treatment group. Dashed lines depict days of therapy administration. Median survival was 15 days for HPBCD vehicle treatment group (n=8), 15 days for AZD4320 treatment group (n=6, p vehicle group, Log-rank test), and 23 days for AZ-MCL1 treatment group (n=13, =0.49 not significant compared with HPBCD p<0.0001 compared with HPBCD vehicle group, Log-rank test).

p p p<0.0001.

NS, not significant; * <0.05, *** <0.001, ****

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a b 60

**** /L ) 60 **** 9 e NS v NS i t i x1 0

40 ( 40 po s

es P

F 20

G 20 %

0 ym pho cy t e 1 0 L e 1 L L ZM C Vehicl ZM C Vehicl A AZD4320 A AZD4320 c

* 8 200 /L ) *** NS 9 g/L ) ( 6 * x1 0 (

100 4 oglobin ophil s r t 2 u ae m e H 0 N 0 e 1 1 0 Vehicl Vehicle AZD4320 AZ-MCL AZ-MCL AZD432 d

1500 **** 100 Vehicle /L )

9 AZ-MCL1 *

1000 va l AZD4320 x1 0 (

rv i

s 50 u t S e

l

e 500 % t a l P 0 0 e 1 0 10 20 30 Days post transplantation Vehicl AZD4320 AZ-MCL

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Figure 5.11: MCL-1 inhibitor therapy is effective against p53 null lymphoma in vivo

(a) Non-irradiated C57BL/6 recipient mice were transplanted by tail vein injection with 1 x 105 Myc #3391 p53-/- lymphoma cells harvested from lymph nodes of a syngeneic mouse. Therapy was commenced three days later with 30% HPBCD Eμ- vehicle or AZ-MCL1 100mg/kg, administered weekly by intravenous tail vein injection for two weeks. A second AZ-MCL1 treatment group received therapy on days three and four post-transplantation, instead of one week apart. (b) Kaplan- Meier curve showing overall survival of mice in each treatment group. Median survival was 18 days for HPBCD vehicle treated mice (n=6), 27 days for AZ-MCL1 treated mice dosed on day three and day 10 (n=9, p<0.0001 Log-rank test compared with vehicle treated group) and 77 days for AZ-MCL1 treated mice dosed on days three and four (n=6, p<0.001 Log-rank test compared with vehicle treated group).

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a Eµ-Myc 1x105 cells

Day 3 Day 4 Day 10

• 30% HPBCD vehicle day 3 & 10 • AZ-MCL1 100mg/kg day 3 & 4 • AZ-MCL1 100mg/kg day 3 & 10

b

100 Vehicle AZMCL1Day 3 & 10 AZMCL1Day 3 & 4 va l

rv i 50 u S

%

0 0 20 40 60 80 100 Days post transplantation

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Figure 5.12: MCL-1 inhibitor therapy is associated with reduced disease progression and prolongation of survival for mice transplanted with human IG-cMYC-translocated Burkitt lymphoma

(a) Non-irradiated NOD-SCID IL2Rγnull immunocompromised recipient mice were transplanted by tail vein injection with 1 x 106 bioluminescence-tagged BL- 41 human Burkitt lymphoma cells. Therapy was commenced three days later with 30% HPBCD vehicle or AZ-MCL1 100mg/kg, administered weekly by intravenous tail vein injection for two weeks. (b) Bioluminescence imaging performed at day seven and day 14 post-transplantation for three representative mice from each housed box. (c) Kaplan-Meier survival curve showing overall survival for mice in each treatment group. Median survival was 19 days for HPBCD vehicle treated group (n=10) and 28 days for AZ-MCL1 treated group (n=10, p<0.0001 Log-rank test).

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a BL-41 1x106 cells

Day 3 Day 10

• 30% HPBCD vehicle day 3 & 10 • AZ-MCL1 100mg/kg day 3 & 10

b Vehicle AZ-MCL1

Day 7

Day 14

c

100 Vehicle AZ-MCL1 va l

rv i 50 u S

%

0 0 10 20 30 40 Days post transplantation

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100 shScrambled ) e shMcl-1.1792 * Base li n (

**

D 50 sR E d %

0 0 1 2 Day

Figure 5.13: Genetic depletion of Mcl-1 with shRNA leads to loss of representation of transduced lymphoma cells

The REBIR.shMcl-1 construct and non-silencing control (shScrambled) were cloned into Eµ-Myc #4242 lymphoma cells and transduced cells were sorted for Eµ-Myc #4242 lymphoma cells transduced with the shMcl-1 construct or shScrambled were exponentially passaged in competitive BFP expression by flow cytometry. culture with non-transduced cells, with or without the addition of doxycycline 1µg/ mL. Flow cytometric analysis of dsRED expression was performed 24 hours post-

p<0.05, addition of doxycycline (arbitrarily defined as day 0) and daily for the next two p<0.005. days. Values are shown normalized to day 0 dsRED positive proportion. * ** Data representative of mean +/- standard error of the mean for three independent experiments.

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5.3 Discussion

5.3.1 MCL-1 inhibition potently induces apoptosis of MYC-driven B-cell lymphoma

As was hypothesised, direct MCL-1 inhibition, but not BCL-2 / BCL-XL inhibition, resulted in rapid induction of apoptosis of MYC-driven murine and human lymphoma cell lines. The dependency of these lymphoma models on MCL-1 was further reinforced by the genetic experiments depleting Mcl-1, and is consistent with recent genetic studies performed by an independent research group (Kelly et al. 2014; Aubrey et al. 2015; Grabow et al. 2016b; 2016a).

It is uncertain why there would be such a distinct difference between forced overexpression of murine Mcl-1 conferring relative protection to Eµ-Myc lymphoma against the MCL-1 inhibitor, while overexpression of Bcl-2 was seen to confer

L inhibition. One possible explanation is that Eµ-Myc lymphoma is inherently sensitive to Mcl-1 inhibition and therefore significant sensitisation to BCL-2 / BCL-X may have an appropriate balance of anti-apoptotic Mcl-1 and pro-apoptotic binding partners in equilibrium, which then shifts to an anti-apoptotic phenotype upon forced overexpression of Mcl-1. In contrast, the inherent insensitivity of Eµ-Myc lymphoma to BCL-2 / BCL-XL inhibition may represent a delicate balance of their expression relative to their cognate pro-apoptotic partners, an equilibrium which is readily unbalanced through forced overexpression of Bcl-2. Such reprogramming of Eµ-Myc lymphoma for dependency to BCL-2 / BCL-XL inhibition with ABT-737 has previously been described by our group and others (Whitecross et al. 2009; Mason et al. 2008).

Another possible explanation for the discordance described above is the highly conserved nature of Bcl-2, as opposed to Mcl-1, and that AZ-MCL1 may possess greater avidity for Homo sapiens versus Mus musculus protein while maintaining in vitro studies (personal communication, Matthew Belmonte and Paul Secrist, specificity. Our collaborators reported similar observations from their preliminary Astra Zeneca, Waltham, MA, USA). This possible explanation is further supported by the in vitro apoptosis assays of AZ-MCL1 treatment of the human Burkitt lymphoma and multiple myeloma cell lines, whereby the LD50 was approximately two doubling dilutions lower than observed in the previous Eµ-Myc studies.

L inhibition is further evidence

Furthermore, the lack of efficacy of BCL-2 / BCL-X for a specific dependency of these models upon MCL-1, rather than non-specific 205 Chapter 5 reduction in the activity of any anti-apoptotic BCL-2 family member. These

L antagonism being ineffective in treatment of Eµ-Myc lymphoma (Mason et al. 2008; Whitecross findings are consistent with the previous report of BCL-2 / BCL-X L inhibitors such as ABT-737, ABT-263 and ABT-199 for treatment of the indolent B-cell et al. 2009), and in contrast to the marked efficacy of BCL-2 / BCL-X lymphoproliferative disorder, CLL (Anderson et al. 2016; Roberts et al. 2016).

5.3.2 MCL-1 inhibition represents a tolerable and effective therapy against MYC-driven B-cell lymphoma in vivo

Despite the observed toxicities of local tail necrosis upon extravasation of AZ- MCL1 at the time of administration, and the hepatic, pancreatic and muscle toxicity detected by serum testing, mice appeared to thrive throughout the AZ-MCL1

bona fide dependency therapy studies and importantly a survival benefit was seen when compared to and therapeutic target in MYC-driven lymphoma, and also demonstrated that a vehicle-treated control mice. This confirms MCL-1 to be a therapeutic window may exist. Despite the potential inter-species variation in sensitivity to MCL-1-inhibition, these therapy studies assessed treatment of Mus musculus lymphoma expressing Mus musculus Mcl-1, indicating the therapeutic window. It would, however, be useful to test this inhibitor in lymphomas derived from and transplanted into humanised MCL1 mice (Zhou et al. 1998; 2001). Clearly, further toxicity testing will be mandated in other models if this compound or analogues are to progress into human clinical trials.

The platelet counts from the same in vivo studies are informative for several reasons. Firstly, no thrombocytopenia was observed from the AZ-MCL1 treated mice, likely representing relatively preserved megakaryopoiesis when compared to the vehicle-treated controls which would have had significant bone marrow phase of lymphoma cells. Secondly, the marked thrombocytopenia observed from infiltration at the time of blood sampling as suggested by the marked leukaemic AZD4320 treatment is consistent with previously published in vivo data using ABT-

737 (Vandenberg and Cory 2013; Souers et al. 2013) and evidence of BCL-XL being the critical determinant of platelet life span (Mason et al. 2007). This provides clear pharmacodynamic evidence of in vivo AZD4320 activity even in the absence in vivo activity of AZD4320 against Eµ-Myc lymphoma in our experiment is consistent with published in vivo studies of efficacy against lymphoma cells. The lack of of ABT-737 (Mason et al. 2008). While neutropenia was observed with AZ-MCL1 treatment in vivo mice and can often be managed supportively in humans. , this toxicity did not lead to any significant observed infections in

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Finally, the dose-schedule in vivo experiment using Eµ-Myc lymphoma #3391 was highly informative in demonstrating curability of MYC-driven lymphoma with just two doses of inhibitor. While this supports the hypothesis of a critical dependency of these lymphomas upon MCL-1, this also demonstrates that dose- dense schedules may be of greater utility when targeting proteins with such short half-lives as MCL-1. Assessment of responses to the dose-dense schedule with further lymphoma clones is warranted in order to support this hypothesis.

5.3.3 Conclusions

MCL-1 has previously provided an elusive therapeutic target, with indirect transcriptional repression being the only historical approach. The experiments in this chapter have supported the hypothesis of MCL-1 as a critical oncogenic dependency of MYC-driven lymphoma. Furthermore, the activity of the selective MCL-1 inhibitor provides the rationale for translation of MCL-1 inhibitor therapies into the clinic.

207 208 Chapter 6: Summation and Conclusions

209 Chapter 6

6.1 The MYC / CDK9 / Pol II / MCL-1 axis as an oncogenic pathway

I hypothesised that MYC recruitment of P-TEFb to target transcriptional sites is a mechanism by which MYC is able to fully activate Pol II through CDK9-mediated phosphorylation of Ser2 at the CTD. Furthermore, historical studies of pan-CDK inhibitors had shown an association between Pol II Ser2 dephosphorylation and reduction in MCL-1 expression in myeloma and CLL cell lines (Gojo et al. 2002; Chen et al. 2005b). As posited, pharmacologic inhibition of CDK9 was associated with reductions in Pol II Ser2 phosphorylation and MCL-1 expression, concomitant pathway and proffer novel therapeutic targets in MYC-driven lymphoma. to apoptosis induction. These findings are supportive of the proposed oncogenic

6.2 Dinaciclib demonstrates marked anti-lymphoma activity

therapeutic potential of this novel CDK inhibitor. Dinaciclib use was hypothesised Studies of dinaciclib described in chapter three demonstrated the significant to effect reductions in Pol II Ser2 phosphorylation and MCL-1 expression and indeed these were observed. Remarkably, short incubations of dinaciclib were seen to potently reduce expression of MYC itself, providing another mechanism of countering the transcriptionally-addicted state of MYC-driven malignancy. The reduction in MCL-1 was clearly not simply an effect of reduced MYC expression, as informative studies of Eµ-Myc lymphoma, where transgenic Myc expression is subject to non-endogenous promoter regulation, demonstrated the same reduction in Mcl-1 with unaffected Myc protein expression.

Dinaciclib studies were also notable for the ability to induce curative responses to mice transplanted with sensitive clones of Eµ-Myc lymphoma. This is remarkable for a targeted approach without chemotherapy and exceeds the activity of any other preclinical compound tested in the Johnstone laboratory previously (Whitecross et al. 2009; Newbold et al. 2013b; Shortt et al. 2013; West et al. 2016). Furthermore, the ability to induce p53-independent apoptosis is of clinical value, as p53 mutation or deletion are frequent events in relapsed and refractory aggressive B-cell lymphomas and confer resistance to conventional chemotherapy and radiotherapy approaches (Ichikawa et al. 1997; Leroy et al. 2002).

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6.2.1 CDK9 inhibitors demonstrate marked anti-lymphoma activity

Dinaciclib has a number of CDK targets, however, the use of additional CDK9 inhibitors in this chapter provided evidence that CDK9 inhibition conferred the biological and therapeutic effects observed with dinaciclib. Furthermore, these inhibitors induced cytostasis to treated cells, the mechanism of which remains unclear. While some CDK inhibitors assessed in this thesis inhibit cell- cycle regulatory CDKs such as CDK1, 2 or 4, transcriptional repression of cyclin expression or other effects of targets such as GSK3 remain as possibly contributory.

6.2.2 Targeting transcriptional addiction in MYC-driven lymphoma

CDK9 inhibition, however, the lentiviral CDK screen demonstrated that opposing the The efficacy of therapeutic agents described in this thesis is largely attributed to activity of other transcriptional CDKs may be of therapeutic value. There remains transcriptional CDKs, particularly CDKs 10, 11, 12, 13, 19 and 20. The marked effects a paucity of studies defining the exact functions and regulation of a number of observed from genetic depletion of CDKs 11 and 13 described herein are supportive of a rationale for further assessment of these potential targets for therapeutic gain. CDK11 represents an interesting target as beyond the kinase function is a possible modulation of Mediator activity (Drogat et al. 2012), and it would be critical to determine whether CDK11 inhibition would lead to destabilisation of Pol II binding as a potent method of inhibition of global transcription. Assessing the effect of CDK11 inhibition on Pol II CTD phosphorylation (Ser2/5), and CDK11-Mediator and Mediator-Pol II binding through co-immunoprecipitation would be useful exploratory experiments to characterise the mechanisms by which antagonism of CDK11 is detrimental to these models in vitro.

Furthermore, while this thesis has provided correlative mechanistic evidence with regard to downstream effects of CDK9 inhibition, it is likely that a number of other effects occur concurrently due to repression of global transcriptional processes. Indeed, the Johnstone Lab is integrating advanced technologies such as global run-on sequencing (Laitem et al. 2015) which will allow for more detailed elucidation of the other effects of CDK9 inhibition such as those upon nascent RNA. These studies will provide insight into the mechanics of the hypothesised transcriptionally-addicted state.

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6.3 MCL-1 inhibition demonstrates marked anti-lymphoma activity

While it was hypothesised that repression of MCL-1 expression was not the only mechanism of anti-lymphoma activity observed with CDK9 inhibition, the MCL-

1 inhibitor studies confirmed the critical oncogenic dependence that MYC-driven recent reports of genetic depletion of MCL1 in murine and human MYC-driven B-cell lymphoma has upon MCL-1. These findings were further supported by the lymphoma models, whereby genetic deletion or depletion of MCL1 reduced the viability of human MYC-driven B-cell lymphoma cell lines and was associated with increased survival of mice when MCL1 dosage was reduced either globally or in B-cell precursor subsets (Kelly et al. 2014; Aubrey et al. 2015; Grabow et al. 2016b; 2016a).

Given the promise recently shown by BCL-2 antagonism in indolent lymphoma such as CLL (Roberts et al. 2016), it will be interesting to observe the responses to MCL-1 inhibitors as they are translated into the clinic for treatment of the aggressive lymphomas. While there has been concern about the toxicities of MCL-1 inhibition due to its broad expression and phenotypic abnormalities upon its deletion, the in vivo studies described herein are supportive of a therapeutic window and marked clinical responses. However, the observed toxicities of local necrosis upon extravasation, as well as pancreatic, hepatic and muscle toxicities certainly warrant cautious investigation.

6.4 Translation of CDK9 inhibitors to the clinic

Following publication of studies described in chapter three in Leukemia, discussions with Merck have led to a phase Ib clinical study of pembrolizumab in combination with dinaciclib in hematologic malignancies (KEYNOTE-155, NCT02684617). Recent data presented at the American Association of Cancer Research Annual Meeting demonstrated dinaciclib treatment to induce immunogenic cell death to synergise with PD-1 blockade in syngeneic mouse tumour models (Hossain et al. 2016). This provided the rationale for the combination of dinaciclib with pembrolizumab in the KEYNOTE-155 clinical trial. It is expected that dinaciclib alone would have single-agent activity against relapsed or refractory DLBCL, and that the addition of pembrolizumab will further prolong responses and perhaps assist in clearance of minimal residual disease. Very pleasingly, Monash Health with DLBCL commenced cycle one of therapy on 14 July 2016. (Victoria, Australia) was chosen as the lead Australian site and the first patient

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There is also rationale for expanding the use of CDK9 inhibition beyond DLBCL. Firstly, dysregulated MYC activity is a hallmark of Burkitt lymphoma and also occurs in other lymphoma subsets (Swerdlow et al. 2008). Due to p53-independent activity, CDK9 inhibition may be of use either upfront or as part of salvage treatment, where loss of functional p53 confers poor prognosis and resistance to other conventional therapies (Ichikawa et al. 1997; Leroy et al. 2002).

Secondly, our group has also recently published on the activity of CDK9 inhibition with dinaciclib as an effective therapy for MLL-driven AML (Baker et al. 2016). to chemotherapy for AML (Zeidner et al. 2015) also warrants further clinical This and another recent publication on the benefit of addition of flavopiridol assessment in this disease area.

6.5 Conclusions

It can be concluded that dinaciclib and other CDK9- and MCL-1-inhibitors represent promising novel therapeutics for the treatment of MYC-driven B-cell lymphoma. Furthermore, the hypothesised transcriptionally-addicted state of MYC-driven disease may enable elucidation of a number of alternative therapeutic strategies with which the oncogenic activity of MYC may be perturbed for therapeutic gain. As these inhibitors are translated into the clinic, assessment of molecular biomarkers combinatorial approaches. will assist stratification of use, as well as resistance mechanisms guiding rational

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Author/s: Gregory, Gareth Peter

Title: Targeting cyclin-dependent kinase 9 and myeloid cell leukaemia 1 in MYC-driven B-cell lymphoma

Date: 2016

Persistent Link: http://hdl.handle.net/11343/123954

File Description: Targeting cyclin-dependent kinase 9 and myeloid cell leukaemia 1 in MYC-driven B-cell lymphoma

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